vero e6 cells  (ATCC)


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    Name:
    VERO C1008 Vero 76 clone E6 Vero E6
    Description:
    Applications VERO C1008 exhibits some degree of contact inhibition after forming a monolayer and is therefore useful in growing slow replicating viruses Host Cercopithecus aethiops
    Catalog Number:
    CRL-1586
    Price:
    None
    Applications:
    VERO C1008 exhibits some degree of contact inhibition after forming a monolayer and is therefore useful in growing slow replicating viruses.
    Host:
    Cercopithecus aethiops
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    Structured Review

    ATCC vero e6 cells
    Pre-treatment of cells with emodin and berberine. Graphic of relative foci number after treatment of <t>Vero</t> <t>E6</t> cells with berberine or emodin. Cells treated with drug diluent were used as negative control. Results are the means (±SD) from three independent experiments and are expressed as relative values compared to untreated cells. *** p
    Applications VERO C1008 exhibits some degree of contact inhibition after forming a monolayer and is therefore useful in growing slow replicating viruses Host Cercopithecus aethiops
    https://www.bioz.com/result/vero e6 cells/product/ATCC
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    vero e6 cells - by Bioz Stars, 2021-06
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    Images

    1) Product Images from "Natural Products Isolated from Oriental Medicinal Herbs Inactivate Zika Virus"

    Article Title: Natural Products Isolated from Oriental Medicinal Herbs Inactivate Zika Virus

    Journal: Viruses

    doi: 10.3390/v11010049

    Pre-treatment of cells with emodin and berberine. Graphic of relative foci number after treatment of Vero E6 cells with berberine or emodin. Cells treated with drug diluent were used as negative control. Results are the means (±SD) from three independent experiments and are expressed as relative values compared to untreated cells. *** p
    Figure Legend Snippet: Pre-treatment of cells with emodin and berberine. Graphic of relative foci number after treatment of Vero E6 cells with berberine or emodin. Cells treated with drug diluent were used as negative control. Results are the means (±SD) from three independent experiments and are expressed as relative values compared to untreated cells. *** p

    Techniques Used: Negative Control

    Viability of emodin- and berberine-treated Vero E6 cells. Cells were incubated with different concentrations of emodin ( A ) or berberine ( B ) and cell viability was evaluated after 24 h, 48 h, and 72 h.
    Figure Legend Snippet: Viability of emodin- and berberine-treated Vero E6 cells. Cells were incubated with different concentrations of emodin ( A ) or berberine ( B ) and cell viability was evaluated after 24 h, 48 h, and 72 h.

    Techniques Used: Incubation

    2) Product Images from "Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis"

    Article Title: Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis

    Journal: Journal of Virology

    doi: 10.1128/JVI.00755-16

    Multistep growth curve of recombinant hMPVs carrying mutations in the phosphorylation site. Vero E6 cells in 35-mm dishes were infected with each recombinant hMPV at an MOI of 0.01. After adsorption for 1 h, the inocula were removed and the infected cells were washed 3 times with Opti-MEM. Fresh Opti-MEM containing 2% FBS then was added and cells were incubated at 37°C for various time periods. Aliquots of the cell culture fluid were removed at the indicated intervals. Viral titer was determined by an immunostaining assay in Vero E6 cells. Viral titers of each mutant at each time point were compared to those of rhMPV (*, P
    Figure Legend Snippet: Multistep growth curve of recombinant hMPVs carrying mutations in the phosphorylation site. Vero E6 cells in 35-mm dishes were infected with each recombinant hMPV at an MOI of 0.01. After adsorption for 1 h, the inocula were removed and the infected cells were washed 3 times with Opti-MEM. Fresh Opti-MEM containing 2% FBS then was added and cells were incubated at 37°C for various time periods. Aliquots of the cell culture fluid were removed at the indicated intervals. Viral titer was determined by an immunostaining assay in Vero E6 cells. Viral titers of each mutant at each time point were compared to those of rhMPV (*, P

    Techniques Used: Recombinant, Infection, Adsorption, Incubation, Cell Culture, Immunostaining, Mutagenesis

    Recovery of recombinant hMPVs carrying mutations in the phosphorylation sites of M2-1. (A) Immunostaining spots formed by recombinant hMPVs. Vero E6 cells were infected with recombinant hMPV mutants and incubated at 37°C for 5 days. The cells were stained with an anti-hMPV N protein monoclonal antibody. (B) Plaque morphology of recombinant hMPVs. An agarose overlay plaque assay was performed on a monolayer of Vero E6 cells. Viral plaques were developed at day 8 postinfection. The plaque pictures were taken from different dilutions of each hMPV mutant. Data were averages from 20 plaques ± 1 standard deviation.
    Figure Legend Snippet: Recovery of recombinant hMPVs carrying mutations in the phosphorylation sites of M2-1. (A) Immunostaining spots formed by recombinant hMPVs. Vero E6 cells were infected with recombinant hMPV mutants and incubated at 37°C for 5 days. The cells were stained with an anti-hMPV N protein monoclonal antibody. (B) Plaque morphology of recombinant hMPVs. An agarose overlay plaque assay was performed on a monolayer of Vero E6 cells. Viral plaques were developed at day 8 postinfection. The plaque pictures were taken from different dilutions of each hMPV mutant. Data were averages from 20 plaques ± 1 standard deviation.

    Techniques Used: Recombinant, Immunostaining, Infection, Incubation, Staining, Plaque Assay, Mutagenesis, Standard Deviation

    3) Product Images from "Successful treatment of Marburg virus with orally administrated T-705 (Favipiravir) in a mouse model"

    Article Title: Successful treatment of Marburg virus with orally administrated T-705 (Favipiravir) in a mouse model

    Journal: Antiviral research

    doi: 10.1016/j.antiviral.2018.01.011

    Antiviral activity of T-705 against MARV in cell culture (A) Vero E6 cells were treated with a dilution series of T-705 (starting at 50 µg/ml followed by 1:3 dilutions) 1 h prior to infection with wild type MARV at MOI of 0.01. After 72 hours, MARV RNA in the supernatant was quantified by RT-qPCR. The EC50 and EC90 values for T-705 with 95% confidence interval (95% CI) were calculated from the sigmoidal function. (B) Virus titers in Vero E6 cell culture supernatants were determined by TCID50 assay.
    Figure Legend Snippet: Antiviral activity of T-705 against MARV in cell culture (A) Vero E6 cells were treated with a dilution series of T-705 (starting at 50 µg/ml followed by 1:3 dilutions) 1 h prior to infection with wild type MARV at MOI of 0.01. After 72 hours, MARV RNA in the supernatant was quantified by RT-qPCR. The EC50 and EC90 values for T-705 with 95% confidence interval (95% CI) were calculated from the sigmoidal function. (B) Virus titers in Vero E6 cell culture supernatants were determined by TCID50 assay.

    Techniques Used: Activity Assay, Cell Culture, Infection, Quantitative RT-PCR, TCID50 Assay

    4) Product Images from "In Vitro and in Vivo Evaluation of Mutations in the NS Region of Lineage 2 West Nile Virus Associated with Neuroinvasiveness in a Mammalian Model"

    Article Title: In Vitro and in Vivo Evaluation of Mutations in the NS Region of Lineage 2 West Nile Virus Associated with Neuroinvasiveness in a Mammalian Model

    Journal: Viruses

    doi: 10.3390/v8020049

    Growth kinetics of infectious virus of the wild type (WT) virus and mutant viruses after triplicate infection of Vero E6 cells at an MOI of 0.1. The titres are given as the mean (log10 TCID 50 /mL); error bars represent standard deviation.
    Figure Legend Snippet: Growth kinetics of infectious virus of the wild type (WT) virus and mutant viruses after triplicate infection of Vero E6 cells at an MOI of 0.1. The titres are given as the mean (log10 TCID 50 /mL); error bars represent standard deviation.

    Techniques Used: Mutagenesis, Infection, Standard Deviation

    Quantification of ( a ) extracellular positive strand RNA ( b ) intracellular positive strand RNA, and ( c ) negative strand RNA for WT and NS1 mutant after triplicate infection of Vero E6 cells at an MOI of 0.1. Copy numbers are given as the mean of two independent experiments (log10 TCID 50 /mL); error bars represent standard deviation.
    Figure Legend Snippet: Quantification of ( a ) extracellular positive strand RNA ( b ) intracellular positive strand RNA, and ( c ) negative strand RNA for WT and NS1 mutant after triplicate infection of Vero E6 cells at an MOI of 0.1. Copy numbers are given as the mean of two independent experiments (log10 TCID 50 /mL); error bars represent standard deviation.

    Techniques Used: Mutagenesis, Infection, Standard Deviation

    5) Product Images from "Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis"

    Article Title: Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis

    Journal: Journal of Virology

    doi: 10.1128/JVI.00755-16

    Multistep growth curve of recombinant hMPVs carrying mutations in the phosphorylation site. Vero E6 cells in 35-mm dishes were infected with each recombinant hMPV at an MOI of 0.01. After adsorption for 1 h, the inocula were removed and the infected cells were washed 3 times with Opti-MEM. Fresh Opti-MEM containing 2% FBS then was added and cells were incubated at 37°C for various time periods. Aliquots of the cell culture fluid were removed at the indicated intervals. Viral titer was determined by an immunostaining assay in Vero E6 cells. Viral titers of each mutant at each time point were compared to those of rhMPV (*, P
    Figure Legend Snippet: Multistep growth curve of recombinant hMPVs carrying mutations in the phosphorylation site. Vero E6 cells in 35-mm dishes were infected with each recombinant hMPV at an MOI of 0.01. After adsorption for 1 h, the inocula were removed and the infected cells were washed 3 times with Opti-MEM. Fresh Opti-MEM containing 2% FBS then was added and cells were incubated at 37°C for various time periods. Aliquots of the cell culture fluid were removed at the indicated intervals. Viral titer was determined by an immunostaining assay in Vero E6 cells. Viral titers of each mutant at each time point were compared to those of rhMPV (*, P

    Techniques Used: Recombinant, Infection, Adsorption, Incubation, Cell Culture, Immunostaining, Mutagenesis

    Recovery of recombinant hMPVs carrying mutations in the phosphorylation sites of M2-1. (A) Immunostaining spots formed by recombinant hMPVs. Vero E6 cells were infected with recombinant hMPV mutants and incubated at 37°C for 5 days. The cells were stained with an anti-hMPV N protein monoclonal antibody. (B) Plaque morphology of recombinant hMPVs. An agarose overlay plaque assay was performed on a monolayer of Vero E6 cells. Viral plaques were developed at day 8 postinfection. The plaque pictures were taken from different dilutions of each hMPV mutant. Data were averages from 20 plaques ± 1 standard deviation.
    Figure Legend Snippet: Recovery of recombinant hMPVs carrying mutations in the phosphorylation sites of M2-1. (A) Immunostaining spots formed by recombinant hMPVs. Vero E6 cells were infected with recombinant hMPV mutants and incubated at 37°C for 5 days. The cells were stained with an anti-hMPV N protein monoclonal antibody. (B) Plaque morphology of recombinant hMPVs. An agarose overlay plaque assay was performed on a monolayer of Vero E6 cells. Viral plaques were developed at day 8 postinfection. The plaque pictures were taken from different dilutions of each hMPV mutant. Data were averages from 20 plaques ± 1 standard deviation.

    Techniques Used: Recombinant, Immunostaining, Infection, Incubation, Staining, Plaque Assay, Mutagenesis, Standard Deviation

    6) Product Images from "Rational Design of Human Metapneumovirus Live Attenuated Vaccine Candidates by Inhibiting Viral mRNA Cap Methyltransferase"

    Article Title: Rational Design of Human Metapneumovirus Live Attenuated Vaccine Candidates by Inhibiting Viral mRNA Cap Methyltransferase

    Journal: Journal of Virology

    doi: 10.1128/JVI.00876-14

    Analysis of hMPV and VSV mRNA cap methylation by in vitro trans -methylation assay. (A) trans -G-N-7 methylation assay for hMPV and VSV. Five hundred nanograms of mRNA was isolated from virus-infected Vero E6 cells and was trans -methylated by vaccinia virus
    Figure Legend Snippet: Analysis of hMPV and VSV mRNA cap methylation by in vitro trans -methylation assay. (A) trans -G-N-7 methylation assay for hMPV and VSV. Five hundred nanograms of mRNA was isolated from virus-infected Vero E6 cells and was trans -methylated by vaccinia virus

    Techniques Used: Methylation, In Vitro, Isolation, Infection

    Sensitivity of recombinant hMPVs to IFN-α (A) and IFN-β (B) treatment. Confluent Vero E6 cells in 24-well plates were pretreated with DMEM containing 2 or 20 U of IFN-α or IFN-β at 37°C for 4 h. Cells were then
    Figure Legend Snippet: Sensitivity of recombinant hMPVs to IFN-α (A) and IFN-β (B) treatment. Confluent Vero E6 cells in 24-well plates were pretreated with DMEM containing 2 or 20 U of IFN-α or IFN-β at 37°C for 4 h. Cells were then

    Techniques Used: Recombinant

    7) Product Images from "Analysis of N-Linked Glycosylation of Hantaan Virus Glycoproteins and the Role of Oligosaccharide Side Chains in Protein Folding and Intracellular Trafficking"

    Article Title: Analysis of N-Linked Glycosylation of Hantaan Virus Glycoproteins and the Role of Oligosaccharide Side Chains in Protein Folding and Intracellular Trafficking

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.10.5414-5422.2004

    Immunoreactivity of wild type (wt) and mutant HTNV glycoproteins with individual anti-Gn and -Gc MAbs. Vero E6 cells were infected with vTF7-3 followed by transfection with wt HTNV M cDNA (pGEM-HTNM) or glycosylation site mutant cDNA as indicated. Cells were then labeled with 50 μCi of [ 35 S]methionine for 15 h and immunoprecipitated with a pair of anti-Gn and anti-Gc MAbs. The resulting immunoprecipitates were analyzed by SDS-10% PAGE under reducing conditions.
    Figure Legend Snippet: Immunoreactivity of wild type (wt) and mutant HTNV glycoproteins with individual anti-Gn and -Gc MAbs. Vero E6 cells were infected with vTF7-3 followed by transfection with wt HTNV M cDNA (pGEM-HTNM) or glycosylation site mutant cDNA as indicated. Cells were then labeled with 50 μCi of [ 35 S]methionine for 15 h and immunoprecipitated with a pair of anti-Gn and anti-Gc MAbs. The resulting immunoprecipitates were analyzed by SDS-10% PAGE under reducing conditions.

    Techniques Used: Mutagenesis, Infection, Transfection, Labeling, Immunoprecipitation, Polyacrylamide Gel Electrophoresis

    Biochemical analyses of N-glycan chains on HTNV glycoproteins Gn and Gc. (A) DNJ blocks N-glycan trimming of Gn and Gc. Vero E6 cells were infected with vT-HTN M and radiolabeled with 50 μCi of [ 35 S]methionine for 15 h in the absence (lanes 1 to 3) or presence (lanes 4 to 6) of 2 mM DNJ, and cell lysates were immunoprecipitated with anti-Gn and -Gc MAbs. The resulting precipitates were subjected to digestion with endo H and PNGase F for 20 h as indicated and analyzed by SDS-10% PAGE under reducing conditions. The deglycosylated forms of Gn and Gc are marked dGn and dGc, respectively. (B) endo H and PNGase F digestion of HTNV glycoproteins labeled with [ 3 H]mannose. Vero E6 cells infected with vT-HTN M were labeled with 200 μCi of [ 3 H]mannose for 4 h, and cell lysates were reacted with anti-Gn and -Gc MAbs, followed by digestion with endo H and PNGase F. The gel was exposed for 60 days. (C) Effect of tunicamycin on glycosylation of HTNV glycoproteins. Vero E6 cells were infected with vTF7-3 and then mock transfected (lane 3) or transfected with HTNV M-segment cDNA tagged with FLAG. Cells were labeled with 50 μCi of [ 35 S]methionine for 15 h in the absence or presence of tunicamycin (2 μg/ml) and immunoprecipitated with anti-Gn and -Gc MAbs (lanes 1 to 3) or anti-FLAG and anti-Gc antibodies (lanes 4 to 7). The immunoprecipitates were subjected to digestion with endo H or PNGase F as indicated.
    Figure Legend Snippet: Biochemical analyses of N-glycan chains on HTNV glycoproteins Gn and Gc. (A) DNJ blocks N-glycan trimming of Gn and Gc. Vero E6 cells were infected with vT-HTN M and radiolabeled with 50 μCi of [ 35 S]methionine for 15 h in the absence (lanes 1 to 3) or presence (lanes 4 to 6) of 2 mM DNJ, and cell lysates were immunoprecipitated with anti-Gn and -Gc MAbs. The resulting precipitates were subjected to digestion with endo H and PNGase F for 20 h as indicated and analyzed by SDS-10% PAGE under reducing conditions. The deglycosylated forms of Gn and Gc are marked dGn and dGc, respectively. (B) endo H and PNGase F digestion of HTNV glycoproteins labeled with [ 3 H]mannose. Vero E6 cells infected with vT-HTN M were labeled with 200 μCi of [ 3 H]mannose for 4 h, and cell lysates were reacted with anti-Gn and -Gc MAbs, followed by digestion with endo H and PNGase F. The gel was exposed for 60 days. (C) Effect of tunicamycin on glycosylation of HTNV glycoproteins. Vero E6 cells were infected with vTF7-3 and then mock transfected (lane 3) or transfected with HTNV M-segment cDNA tagged with FLAG. Cells were labeled with 50 μCi of [ 35 S]methionine for 15 h in the absence or presence of tunicamycin (2 μg/ml) and immunoprecipitated with anti-Gn and -Gc MAbs (lanes 1 to 3) or anti-FLAG and anti-Gc antibodies (lanes 4 to 7). The immunoprecipitates were subjected to digestion with endo H or PNGase F as indicated.

    Techniques Used: Infection, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Labeling, Transfection

    Association of HTNV glycoproteins with CNX and CRT. Vero E6 cells were infected with vTF7-3 followed by transfection with wild-type or N glycosylation site mutant HTNV M cDNAs and then labeled for 30 min with 50 μCi of [ 35 S]methionine in the absence (A and B, lanes 1, 3, 5, and 7; C, lanes 1 to 4) or presence (A and B, lanes 2, 4, 6, and 8) of DNJ. Cell lysates were divided into three aliquots for immunoprecipitation with anti-Gn/Gc (A), anti-CNX (B), or anti-CRT (C) antibodies. The resulting immunoprecipitates were analyzed by SDS-10% PAGE under reducing conditions.
    Figure Legend Snippet: Association of HTNV glycoproteins with CNX and CRT. Vero E6 cells were infected with vTF7-3 followed by transfection with wild-type or N glycosylation site mutant HTNV M cDNAs and then labeled for 30 min with 50 μCi of [ 35 S]methionine in the absence (A and B, lanes 1, 3, 5, and 7; C, lanes 1 to 4) or presence (A and B, lanes 2, 4, 6, and 8) of DNJ. Cell lysates were divided into three aliquots for immunoprecipitation with anti-Gn/Gc (A), anti-CNX (B), or anti-CRT (C) antibodies. The resulting immunoprecipitates were analyzed by SDS-10% PAGE under reducing conditions.

    Techniques Used: Infection, Transfection, Mutagenesis, Labeling, Immunoprecipitation, Polyacrylamide Gel Electrophoresis

    Expression of single N glycosylation site mutants of HTNV glycoproteins. Vero E6 cells were infected with vTF7-3 followed by transfection with wild type (Wt) or mutated HTNV M cDNA. Cells were labeled with 50 μCi of [ 35 S]methionine for 15 h and then immunoprecipitated with anti-Gn and -Gc MAbs. Immunoprecipitates were analyzed by SDS-10% PAGE under reducing conditions.
    Figure Legend Snippet: Expression of single N glycosylation site mutants of HTNV glycoproteins. Vero E6 cells were infected with vTF7-3 followed by transfection with wild type (Wt) or mutated HTNV M cDNA. Cells were labeled with 50 μCi of [ 35 S]methionine for 15 h and then immunoprecipitated with anti-Gn and -Gc MAbs. Immunoprecipitates were analyzed by SDS-10% PAGE under reducing conditions.

    Techniques Used: Expressing, Infection, Transfection, Labeling, Immunoprecipitation, Polyacrylamide Gel Electrophoresis

    Pulse-chase analysis of wild type (wt) and three N glycosylation site mutant glycoproteins. Vero E6 cells were infected with vTF7-3 followed by transfection with wt or mutant cDNA, labeled for 20 min with 80 μCi of [ 35 S]methionine (200 μCi/ml), and then incubated with excess unlabeled methionine for the indicated times. Cell lysates were immunoprecipitated with anti-Gn MAb 16D2 and two anti-Gc MAbs, 11E10 and HCO2. The resulting immunoprecipitates were analyzed by SDS-10% PAGE under reducing conditions (A) or by SDS-8% PAGE under nonreducing conditions (B).
    Figure Legend Snippet: Pulse-chase analysis of wild type (wt) and three N glycosylation site mutant glycoproteins. Vero E6 cells were infected with vTF7-3 followed by transfection with wt or mutant cDNA, labeled for 20 min with 80 μCi of [ 35 S]methionine (200 μCi/ml), and then incubated with excess unlabeled methionine for the indicated times. Cell lysates were immunoprecipitated with anti-Gn MAb 16D2 and two anti-Gc MAbs, 11E10 and HCO2. The resulting immunoprecipitates were analyzed by SDS-10% PAGE under reducing conditions (A) or by SDS-8% PAGE under nonreducing conditions (B).

    Techniques Used: Pulse Chase, Mutagenesis, Infection, Transfection, Labeling, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis

    8) Product Images from "Live imaging of SARS-CoV-2 infection in mice reveals neutralizing antibodies require Fc function for optimal efficacy"

    Article Title: Live imaging of SARS-CoV-2 infection in mice reveals neutralizing antibodies require Fc function for optimal efficacy

    Journal: bioRxiv

    doi: 10.1101/2021.03.22.436337

    EM localization of SARS-CoV-2 Virions in Lung, Brain and Testis of Infected K18-hACE2 Mice. (A) 2D overview of a lung region featuring red blood cells (rbc) within a pulmonary capillary, an alveolar Type 2 cell (AT2). (B) Slice from a 3D tomogram of square region in A showing membrane-enclosed cytoplasmic compartments (arrowheads) containing presumptive SARS-CoV-2 virions in capillary endothelial cells. (C) Presumptive virions from tomogram in B displayed at equatorial views. Presumptive virions were identified as described in the Methods and are directly comparable to those in SARS-CoV-2 infected Vero-E6 cells (panels O-Q). (D) ImmunoEM tomography of presumptive SARS-CoV-2 virions from infected lung tissue, labeled with antiserum against Spike protein and gold (10 nm) conjugated 2° antibodies. Gold particles localized to the outer peripheries of the virions indicate specific labeling of SARS-CoV-2 Spikes. (E) Tomographic of SARS-CoV-2 infected brain tissue. Presumptive SARS-CoV-2 virions (red arrowheads) are present within a neuron (pale green). A dendritic synaptic terminal to the left of the virus-containing neuron shows that presumptive SARS-CoV-2 virions are easily distinguished from typical synaptic neurotransmitter vesicles. (F) 2D overview of brain tissue illustrating the complex spatial relationship among neurons and other brain cell types. Presumptive SARS-CoV-2 virions are present in two compartments (black squares) within a single neuron. (G, H) Tomographic slices of black squares in F. Presumptive SARS-CoV-2 virions (red arrowheads) appear to be aligned within compartments that border the edges of a neural projection. (I) Presumptive SARS-CoV-2 virions from tomograms in G and H. (J) ImmunoEM tomography as in D of presumptive SARS-CoV-2 virions from infected brain tissue. (K) (Upper) BLI of testis from a SARS-CoV-2 infected mouse to identify infected regions for IF and EM analyses. (Lower) IF image of an infected testis region stained with antibodies to SARS-CoV-2 Nucleocapsid (red) (L) 2D overview of testis corresponding to region of high intensity (red) in the upper panel of K, showing Sertoli cells surrounded by developing sperm (left) and one primary spermatocyte (1°S, upper right). Presumptive SARS-CoV-2 virions are localized to membrane-bound compartments in Sertoli cells (black squares). (M, N) Slices from two 3D tomograms of squares in L. Presumptive SARS-CoV-2 virions (arrowheads) are present within membrane-enclosed cytoplasmic compartments. These compartments contain additional structures amongst the discernable SARS-CoV-2 virions (insets). (O) EM localization of virions in SARS-CoV-2 infected Vero-E6 cells, processed for EM as above tissue samples. Virions were characterized (see Methods) and compared to presumptive virions in the tissue samples to confidently verify their identities. 2D overview of infected Vero-E6 cell in a 150 nm section. (P) Tomogram of rectangle in O showing > 100 presumptive SARS-CoV-2 virions contained within cytoplasmic exit compartments. (Q) Virions from the tomogram in P showing common features of dense RNC puncta, discernable surface spikes, vary in size (∼60-120 nm) and shape. Virions are directly comparable to those shown for the tissue samples in C and I.
    Figure Legend Snippet: EM localization of SARS-CoV-2 Virions in Lung, Brain and Testis of Infected K18-hACE2 Mice. (A) 2D overview of a lung region featuring red blood cells (rbc) within a pulmonary capillary, an alveolar Type 2 cell (AT2). (B) Slice from a 3D tomogram of square region in A showing membrane-enclosed cytoplasmic compartments (arrowheads) containing presumptive SARS-CoV-2 virions in capillary endothelial cells. (C) Presumptive virions from tomogram in B displayed at equatorial views. Presumptive virions were identified as described in the Methods and are directly comparable to those in SARS-CoV-2 infected Vero-E6 cells (panels O-Q). (D) ImmunoEM tomography of presumptive SARS-CoV-2 virions from infected lung tissue, labeled with antiserum against Spike protein and gold (10 nm) conjugated 2° antibodies. Gold particles localized to the outer peripheries of the virions indicate specific labeling of SARS-CoV-2 Spikes. (E) Tomographic of SARS-CoV-2 infected brain tissue. Presumptive SARS-CoV-2 virions (red arrowheads) are present within a neuron (pale green). A dendritic synaptic terminal to the left of the virus-containing neuron shows that presumptive SARS-CoV-2 virions are easily distinguished from typical synaptic neurotransmitter vesicles. (F) 2D overview of brain tissue illustrating the complex spatial relationship among neurons and other brain cell types. Presumptive SARS-CoV-2 virions are present in two compartments (black squares) within a single neuron. (G, H) Tomographic slices of black squares in F. Presumptive SARS-CoV-2 virions (red arrowheads) appear to be aligned within compartments that border the edges of a neural projection. (I) Presumptive SARS-CoV-2 virions from tomograms in G and H. (J) ImmunoEM tomography as in D of presumptive SARS-CoV-2 virions from infected brain tissue. (K) (Upper) BLI of testis from a SARS-CoV-2 infected mouse to identify infected regions for IF and EM analyses. (Lower) IF image of an infected testis region stained with antibodies to SARS-CoV-2 Nucleocapsid (red) (L) 2D overview of testis corresponding to region of high intensity (red) in the upper panel of K, showing Sertoli cells surrounded by developing sperm (left) and one primary spermatocyte (1°S, upper right). Presumptive SARS-CoV-2 virions are localized to membrane-bound compartments in Sertoli cells (black squares). (M, N) Slices from two 3D tomograms of squares in L. Presumptive SARS-CoV-2 virions (arrowheads) are present within membrane-enclosed cytoplasmic compartments. These compartments contain additional structures amongst the discernable SARS-CoV-2 virions (insets). (O) EM localization of virions in SARS-CoV-2 infected Vero-E6 cells, processed for EM as above tissue samples. Virions were characterized (see Methods) and compared to presumptive virions in the tissue samples to confidently verify their identities. 2D overview of infected Vero-E6 cell in a 150 nm section. (P) Tomogram of rectangle in O showing > 100 presumptive SARS-CoV-2 virions contained within cytoplasmic exit compartments. (Q) Virions from the tomogram in P showing common features of dense RNC puncta, discernable surface spikes, vary in size (∼60-120 nm) and shape. Virions are directly comparable to those shown for the tissue samples in C and I.

    Techniques Used: Infection, Mouse Assay, Labeling, Staining

    Monocytes, Neutrophils and Natural Killer Cells Contribute to Antibody Effector Functions In Vivo . (A) Experimental design to test the contribution of NK cells, neutrophils (CD11b + Ly6G + ) and monocytes (CCR2 + Ly6 hi CD11b + ) in K18-hACE2 mice therapeutically treated with CV3-1 NAb (i.p.,12.5 mg/kg body weight) at 3 dpi after challenge with SARS-CoV-2-nLuc. αNK1.1 mAb (i.p., 20 mg/kg body weight), αLy6G mAb (i.p., 20 mg/kg body weight) and αCCR2 mAb (i.p., 2.5 mg/kg body weight) were used to deplete NK cells, neutrophils and monocytes respectively every 48h starting at 1 dpi. Corresponding human (for CV3-1) and rat (for αNK1.1 and αLy6G mAb or αCCR2) monoclonal antibodies served as non-specific isotype controls (Iso). The mice were followed by non-invasive BLI every 2 days from the start of infection. (B) Representative images from temporal BLI of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions at the indicated dpi and after necropsy. (C-D) Temporal quantification of nLuc signal acquired non-invasively as flux (photons/sec) in indicated regions. (E) Temporal changes in mouse body weight at indicated dpi with initial body weight set to 100%. (F) Kaplan-Meier survival curves of mice statistically compared by log-rank (Mantel-Cox) test for experiment as in A. (H) Viral loads (nLuc activity/g) of indicated organs using Vero E6 cells as targets. Undetectable virus amounts were set to 1. (I, J) Cytokine mRNA levels in lung and brain tissues after necropsy and normalization to GAPDH in the same sample and that in uninfected mice. Viral loads (H) and inflammatory cytokine profile (I, J) was determined after necropsy at times indicated in B. Each curve in C-E and each data point in H-J represents an individual mouse. Grouped data in (C)-(I) were analyzed by 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests. Statistical significance: group comparisons to isotype control are shown in black; group comparisons to Iso+CV3-1 within the NK and neutrophil depleted cohorts are shown in purple; group comparisons to Iso+CV3-1 within the monocyte-depleted cohorts are shown in red. ∗, p
    Figure Legend Snippet: Monocytes, Neutrophils and Natural Killer Cells Contribute to Antibody Effector Functions In Vivo . (A) Experimental design to test the contribution of NK cells, neutrophils (CD11b + Ly6G + ) and monocytes (CCR2 + Ly6 hi CD11b + ) in K18-hACE2 mice therapeutically treated with CV3-1 NAb (i.p.,12.5 mg/kg body weight) at 3 dpi after challenge with SARS-CoV-2-nLuc. αNK1.1 mAb (i.p., 20 mg/kg body weight), αLy6G mAb (i.p., 20 mg/kg body weight) and αCCR2 mAb (i.p., 2.5 mg/kg body weight) were used to deplete NK cells, neutrophils and monocytes respectively every 48h starting at 1 dpi. Corresponding human (for CV3-1) and rat (for αNK1.1 and αLy6G mAb or αCCR2) monoclonal antibodies served as non-specific isotype controls (Iso). The mice were followed by non-invasive BLI every 2 days from the start of infection. (B) Representative images from temporal BLI of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions at the indicated dpi and after necropsy. (C-D) Temporal quantification of nLuc signal acquired non-invasively as flux (photons/sec) in indicated regions. (E) Temporal changes in mouse body weight at indicated dpi with initial body weight set to 100%. (F) Kaplan-Meier survival curves of mice statistically compared by log-rank (Mantel-Cox) test for experiment as in A. (H) Viral loads (nLuc activity/g) of indicated organs using Vero E6 cells as targets. Undetectable virus amounts were set to 1. (I, J) Cytokine mRNA levels in lung and brain tissues after necropsy and normalization to GAPDH in the same sample and that in uninfected mice. Viral loads (H) and inflammatory cytokine profile (I, J) was determined after necropsy at times indicated in B. Each curve in C-E and each data point in H-J represents an individual mouse. Grouped data in (C)-(I) were analyzed by 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests. Statistical significance: group comparisons to isotype control are shown in black; group comparisons to Iso+CV3-1 within the NK and neutrophil depleted cohorts are shown in purple; group comparisons to Iso+CV3-1 within the monocyte-depleted cohorts are shown in red. ∗, p

    Techniques Used: In Vivo, Mouse Assay, Infection, Activity Assay

    CV3-1 Therapy Protects Mice from Lethal SARS-CoV-2 Infection. (A) Experimental design to test in vivo efficacy of CV3-1 administered i.p. (12.5 mg/kg body weight) at indicated times after i.n. challenge of K18-hACE2 mice with SARS-CoV-2 nLuc followed by non-invasive BLI every 2 days. Human IgG1 treated (12.5 mg/kg body weight) mice were the control cohort. (B) Representative images from temporal BLI of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions at the indicated dpi and after necropsy. (C-D) Temporal quantification of nLuc signal acquired non-invasively as flux (photons/sec) in indicated regions of each mice. (E) Temporal changes in mouse body weight at indicated dpi with initial body weight set to 100%. (F) Kaplan-Meier survival curves of mice statistically compared by log-rank (Mantel-Cox) test for experiment as in A. (G, H) Ex vivo imaging of indicated organs and quantification of nLuc signal as flux(photons/sec) at indicated dpi after necropsy. (I) Viral loads estimated as nLuc activity/g of indicated organs using Vero E6 cells as targets. Non-detectable virus amounts were set to 1. (J, K) Cytokine mRNA levels in lung and brain tissues after necropsy. The data was normalized to Gapdh in the same sample and that in uninfected mice. Viral loads (I) and inflammatory cytokine profile (J, K) were determined after necropsy at times indicated in G. Each curve in (C)-(E) and each data point in (H)-(K) represents an individual mouse. CV3-1 treatment times are indicated in (C)-(E). Grouped data in (C)-(K) were analyzed by 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests. Statistical significance for group comparisons to isotype control are shown in black and for those under CV3-1 therapies to 4 dpi-treated cohorts are shown in red. ∗, p
    Figure Legend Snippet: CV3-1 Therapy Protects Mice from Lethal SARS-CoV-2 Infection. (A) Experimental design to test in vivo efficacy of CV3-1 administered i.p. (12.5 mg/kg body weight) at indicated times after i.n. challenge of K18-hACE2 mice with SARS-CoV-2 nLuc followed by non-invasive BLI every 2 days. Human IgG1 treated (12.5 mg/kg body weight) mice were the control cohort. (B) Representative images from temporal BLI of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions at the indicated dpi and after necropsy. (C-D) Temporal quantification of nLuc signal acquired non-invasively as flux (photons/sec) in indicated regions of each mice. (E) Temporal changes in mouse body weight at indicated dpi with initial body weight set to 100%. (F) Kaplan-Meier survival curves of mice statistically compared by log-rank (Mantel-Cox) test for experiment as in A. (G, H) Ex vivo imaging of indicated organs and quantification of nLuc signal as flux(photons/sec) at indicated dpi after necropsy. (I) Viral loads estimated as nLuc activity/g of indicated organs using Vero E6 cells as targets. Non-detectable virus amounts were set to 1. (J, K) Cytokine mRNA levels in lung and brain tissues after necropsy. The data was normalized to Gapdh in the same sample and that in uninfected mice. Viral loads (I) and inflammatory cytokine profile (J, K) were determined after necropsy at times indicated in G. Each curve in (C)-(E) and each data point in (H)-(K) represents an individual mouse. CV3-1 treatment times are indicated in (C)-(E). Grouped data in (C)-(K) were analyzed by 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests. Statistical significance for group comparisons to isotype control are shown in black and for those under CV3-1 therapies to 4 dpi-treated cohorts are shown in red. ∗, p

    Techniques Used: Mouse Assay, Infection, In Vivo, Ex Vivo, Imaging, Activity Assay

    In vitro Characterization of CV3-1 and CV3-25 NAbs. (A) NAb binding to SARS-CoV-2 Spike ectodomain (S-6P) or RBD estimated by ELISA. Relative light unit (RLU) were normalized to the cross-reactive SARS-CoV-1 NAb CR3022. NAb binding to SARS-CoV-2 S2 N-His protein on cell-surface of transfected 293T cells analyzed by flow cytometry. Median fluorescence intensities (MFIs) for anti-Spike NAbs were normalized to the signal obtained with an anti-His tag mAb. (B) Flow cytometric detection of S-expressing 293T cells from the different human CoVs: SARS-CoV-2, SARS-CoV-1, OC43, HKU1, MERS-CoV, NL63 and 229E. MFI from 293T cells transfected with empty vector were used for normalization. (C) Pseudoviruses bearing SARS-CoV-2 or SARS-CoV-1 S were tested for capture by anti-Spike NAbs. The cross-reactive CR3022 mAb was used for normalization. (D-E) NAb binding affinity and kinetics to SARS-CoV-2 S using Surface Plasmon Resonance (SPR). SARS-CoV-2 S-6P or S2 ectodomain was immobilized as the ligand on the chip and CV3-1 or CV3-25 Fab was used as analytes at concentrations in a range from 1.56 to 100 nM for both Fabs to S-6P and 3.125nM to 200nM for CV3-25 to S2 (2-fold serial dilution, see Methods for details). Alternatively, CV3-1 IgG was immobilized on the chip and SARS-CoV-2 RBD was used as the analyte with concentrations ranging from 1.56 to 50 nM (2-fold serial dilution). Kinetic constants were determined using a 1:1 Langmuir model in BIA evaluation software (experimental readings shown in blue and fitted curves shown in black). (F-H) FRET histograms of ligand-free S on S-MEN coronavirus-like particles (VLPs) or in presence of 50 µg/mL of CV3-1 (G) or CV3-25 (H). VLPs were incubated for 1 h at 37°C before smFRET imaging. N m is the number of individual FRET traces compiled into a conformation-population FRET histogram (gray lines) and fitted into a 4-state Gaussian distribution (solid black) centered at 0.1-FRET (dashed cyan), 0.3-FRET (dashed red), 0.5-FRET (dashed green), and 0.8-FRET (dashed magenta). (I) Neutralizing activity of CV3-1 and CV3-25 alone or in combination (1:1 ratio) on SARS-CoV-2 S bearing pseudoviruses using 293T-ACE2 cells. (J) Microneutralization activity of anti-Spike NAbs on live SARS-CoV-2 virus using Vero E6 cells. (K) Inhibition of cell-to-cell fusion between 293T cells expressing HIV-1 Tat and SARS-CoV-2 S and TZM-bl-ACE2 cells by NAbs. Half maximal inhibitory antibody concentration (IC 50 ) values in I-K were determined by normalized non-linear regression analyses. (L) MFI of CEM.NKr cells expressing SARS-CoV-2 Spike (CEM.NKr-Spike) stained with indicated amounts of NAbs and normalized to parental CEM.NKr. (M) % ADCC in the presence of titrated amounts of NAbs using 1:1 ratio of parental CEM.NKr cells and CEM.NKr-Spike cells as targets when PBMCs from uninfected donors were used as effector cells (N) % ADCP in the presence of titrated amounts of NAbs using CEM.NKr-Spike cells as targets and THP-1 cells as phagocytic cells.
    Figure Legend Snippet: In vitro Characterization of CV3-1 and CV3-25 NAbs. (A) NAb binding to SARS-CoV-2 Spike ectodomain (S-6P) or RBD estimated by ELISA. Relative light unit (RLU) were normalized to the cross-reactive SARS-CoV-1 NAb CR3022. NAb binding to SARS-CoV-2 S2 N-His protein on cell-surface of transfected 293T cells analyzed by flow cytometry. Median fluorescence intensities (MFIs) for anti-Spike NAbs were normalized to the signal obtained with an anti-His tag mAb. (B) Flow cytometric detection of S-expressing 293T cells from the different human CoVs: SARS-CoV-2, SARS-CoV-1, OC43, HKU1, MERS-CoV, NL63 and 229E. MFI from 293T cells transfected with empty vector were used for normalization. (C) Pseudoviruses bearing SARS-CoV-2 or SARS-CoV-1 S were tested for capture by anti-Spike NAbs. The cross-reactive CR3022 mAb was used for normalization. (D-E) NAb binding affinity and kinetics to SARS-CoV-2 S using Surface Plasmon Resonance (SPR). SARS-CoV-2 S-6P or S2 ectodomain was immobilized as the ligand on the chip and CV3-1 or CV3-25 Fab was used as analytes at concentrations in a range from 1.56 to 100 nM for both Fabs to S-6P and 3.125nM to 200nM for CV3-25 to S2 (2-fold serial dilution, see Methods for details). Alternatively, CV3-1 IgG was immobilized on the chip and SARS-CoV-2 RBD was used as the analyte with concentrations ranging from 1.56 to 50 nM (2-fold serial dilution). Kinetic constants were determined using a 1:1 Langmuir model in BIA evaluation software (experimental readings shown in blue and fitted curves shown in black). (F-H) FRET histograms of ligand-free S on S-MEN coronavirus-like particles (VLPs) or in presence of 50 µg/mL of CV3-1 (G) or CV3-25 (H). VLPs were incubated for 1 h at 37°C before smFRET imaging. N m is the number of individual FRET traces compiled into a conformation-population FRET histogram (gray lines) and fitted into a 4-state Gaussian distribution (solid black) centered at 0.1-FRET (dashed cyan), 0.3-FRET (dashed red), 0.5-FRET (dashed green), and 0.8-FRET (dashed magenta). (I) Neutralizing activity of CV3-1 and CV3-25 alone or in combination (1:1 ratio) on SARS-CoV-2 S bearing pseudoviruses using 293T-ACE2 cells. (J) Microneutralization activity of anti-Spike NAbs on live SARS-CoV-2 virus using Vero E6 cells. (K) Inhibition of cell-to-cell fusion between 293T cells expressing HIV-1 Tat and SARS-CoV-2 S and TZM-bl-ACE2 cells by NAbs. Half maximal inhibitory antibody concentration (IC 50 ) values in I-K were determined by normalized non-linear regression analyses. (L) MFI of CEM.NKr cells expressing SARS-CoV-2 Spike (CEM.NKr-Spike) stained with indicated amounts of NAbs and normalized to parental CEM.NKr. (M) % ADCC in the presence of titrated amounts of NAbs using 1:1 ratio of parental CEM.NKr cells and CEM.NKr-Spike cells as targets when PBMCs from uninfected donors were used as effector cells (N) % ADCP in the presence of titrated amounts of NAbs using CEM.NKr-Spike cells as targets and THP-1 cells as phagocytic cells.

    Techniques Used: In Vitro, Binding Assay, Enzyme-linked Immunosorbent Assay, Transfection, Flow Cytometry, Fluorescence, Expressing, Plasmid Preparation, SPR Assay, Chromatin Immunoprecipitation, Serial Dilution, Software, Incubation, Imaging, Activity Assay, Inhibition, Concentration Assay, Staining

    Prophylactic Treatment with CV3-1 Protects Mice from Lethal SARS-CoV-2 Infection. (A) Experimental design for testing in vivo efficacy of NAbs CV3-1 and CV3-25 administered alone (12.5 mg/kg body weight) or as a 1:1 cocktail (6.25 mg/kg body weight each) 1 day prior to challenging K18-hACE2 mice (i.n.) with SARS-CoV-2-nLuc followed by non-invasive BLI every 2 days. Human IgG1-treated (12.5 mg Ig/kg) mice were use as the isotype control (Iso) (B) Representative images from BLI of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions at the indicated dpi and after necropsy at indicated days for experiment as in A. (C-D) Temporal quantification of nLuc signal as flux (photons/sec) computed non-invasively in indicated areas of each animal. (E) Temporal changes in mouse body weight at the indicated dpi with initial body weight set to 100%. (F) Kaplan-Meier survival curves of mice statistically compared by log-rank (Mantel-Cox) test for experiment as in A. (G, H) Ex-vivo imaging of organs and quantification of nLuc signal as flux (photons/sec) at the indicated dpi after necropsy. (I) Viral loads (nLuc activity/g) from indicated organs using Vero E6 cells as targets. Undetectable virus amounts were set to 1. (J, K) Cytokine mRNA levels in lung and brain tissues after necropsy normalized to Gapdh in the same sample and that in uninfected mice. Viral loads (I) and inflammatory cytokine profile (J, K) were determined after necropsy for mice that succumbed to infection and in mice surviving at 22 dpi. Scale bars in (B) and (G) denote radiance (photons/sec/cm 2 /steradian). Each curve in (C)-(E) and each data point in (H)-(K) represents an individual mouse. Grouped data in (C)-(K) were analyzed by 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests. Statistical significance for group comparisons to isotype control are shown in black and for those to CV3-25 are shown in red ∗, p
    Figure Legend Snippet: Prophylactic Treatment with CV3-1 Protects Mice from Lethal SARS-CoV-2 Infection. (A) Experimental design for testing in vivo efficacy of NAbs CV3-1 and CV3-25 administered alone (12.5 mg/kg body weight) or as a 1:1 cocktail (6.25 mg/kg body weight each) 1 day prior to challenging K18-hACE2 mice (i.n.) with SARS-CoV-2-nLuc followed by non-invasive BLI every 2 days. Human IgG1-treated (12.5 mg Ig/kg) mice were use as the isotype control (Iso) (B) Representative images from BLI of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions at the indicated dpi and after necropsy at indicated days for experiment as in A. (C-D) Temporal quantification of nLuc signal as flux (photons/sec) computed non-invasively in indicated areas of each animal. (E) Temporal changes in mouse body weight at the indicated dpi with initial body weight set to 100%. (F) Kaplan-Meier survival curves of mice statistically compared by log-rank (Mantel-Cox) test for experiment as in A. (G, H) Ex-vivo imaging of organs and quantification of nLuc signal as flux (photons/sec) at the indicated dpi after necropsy. (I) Viral loads (nLuc activity/g) from indicated organs using Vero E6 cells as targets. Undetectable virus amounts were set to 1. (J, K) Cytokine mRNA levels in lung and brain tissues after necropsy normalized to Gapdh in the same sample and that in uninfected mice. Viral loads (I) and inflammatory cytokine profile (J, K) were determined after necropsy for mice that succumbed to infection and in mice surviving at 22 dpi. Scale bars in (B) and (G) denote radiance (photons/sec/cm 2 /steradian). Each curve in (C)-(E) and each data point in (H)-(K) represents an individual mouse. Grouped data in (C)-(K) were analyzed by 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests. Statistical significance for group comparisons to isotype control are shown in black and for those to CV3-25 are shown in red ∗, p

    Techniques Used: Mouse Assay, Infection, In Vivo, Ex Vivo, Imaging, Activity Assay

    Visualization of SARS-CoV-2 Replication Dynamics in hACE2 Transgenic Mice (A) Experimental strategy utilizing SARS-CoV-2 carrying nLuc reporter in ORF7 for non-invasive BLI of virus spread following intranasal (i.n.) challenge of B6 or K18-hACE2 mice. (B) Representative images from temporal BLI of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions at the indicated dpi and after necropsy at 6 dpi. (C) Temporal quantification of nLuc signal as flux (photons/sec) acquired non-invasively in the indicated tissues of each animal. The color bar above the x-axis (yellow to orange) represents computed signal intensities in K18-hACE2 mice that are significantly above those in B6 mice. (D) Temporal changes in mouse body weight at the indicated dpi with initial body weight set to 100%. (E) Kaplan-Meier survival curves of mice for experiment as in A statistically compared by log-rank (Mantel-Cox) test. (F) Ex vivo imaging of indicated organs and quantification of nLuc signal as flux(photons/sec) at indicated dpi after necropsy (G, H) Viral loads (FFUs/g or nLuc activity/g) from indicated tissue using Vero E6 cells as targets. Undetectable virus amounts were set to 1. (I) Ratio of C t values for SARS-CoV-2 nucleocapsid (N) and nLuc estimated by RT-PCR using RNA extracted from input virions (inoculum) and virions from sera of mice at 6 dpi. (J, K) Cytokine mRNA levels in lung and brain tissues at 6 dpi after normalization to Gapdh in the same sample and that in uninfected mice. Each curve in (C) and (D) and each data point in (F), (I), (J), and (K) represents an individual mouse. Scale bars in (B and (F) denote radiance (photons/sec/cm 2 /steradian). p values obtained by non-parametric Mann-Whitney test for pairwise comparison. ∗, p
    Figure Legend Snippet: Visualization of SARS-CoV-2 Replication Dynamics in hACE2 Transgenic Mice (A) Experimental strategy utilizing SARS-CoV-2 carrying nLuc reporter in ORF7 for non-invasive BLI of virus spread following intranasal (i.n.) challenge of B6 or K18-hACE2 mice. (B) Representative images from temporal BLI of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions at the indicated dpi and after necropsy at 6 dpi. (C) Temporal quantification of nLuc signal as flux (photons/sec) acquired non-invasively in the indicated tissues of each animal. The color bar above the x-axis (yellow to orange) represents computed signal intensities in K18-hACE2 mice that are significantly above those in B6 mice. (D) Temporal changes in mouse body weight at the indicated dpi with initial body weight set to 100%. (E) Kaplan-Meier survival curves of mice for experiment as in A statistically compared by log-rank (Mantel-Cox) test. (F) Ex vivo imaging of indicated organs and quantification of nLuc signal as flux(photons/sec) at indicated dpi after necropsy (G, H) Viral loads (FFUs/g or nLuc activity/g) from indicated tissue using Vero E6 cells as targets. Undetectable virus amounts were set to 1. (I) Ratio of C t values for SARS-CoV-2 nucleocapsid (N) and nLuc estimated by RT-PCR using RNA extracted from input virions (inoculum) and virions from sera of mice at 6 dpi. (J, K) Cytokine mRNA levels in lung and brain tissues at 6 dpi after normalization to Gapdh in the same sample and that in uninfected mice. Each curve in (C) and (D) and each data point in (F), (I), (J), and (K) represents an individual mouse. Scale bars in (B and (F) denote radiance (photons/sec/cm 2 /steradian). p values obtained by non-parametric Mann-Whitney test for pairwise comparison. ∗, p

    Techniques Used: Transgenic Assay, Mouse Assay, Infection, Ex Vivo, Imaging, Activity Assay, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

    Fc-mediated Antibody Effector Functions Contribute to the In Vivo Efficacy of CV3-1 (A) Experimental design to test therapeutic efficacy of NAb CV3-1 and its corresponding Leucine to Alanine (LALA) mutant administered ip (12.5 mg/kg body weight) 3 dpi to K18-hACE2 mice challenged with SARS-CoV-2 nLuc followed by non-invasive BLI every 2 days. Human IgG1-treated (12.5 mg/kg body weight) mice were used as the control cohort. (B) Representative images from temporal BLI of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions at the indicated dpi and after necropsy. Scale bars denote radiance (photons/sec/cm 2 /steradian). (C-D) Temporal quantification of nLuc signal acquired non-invasively as flux (photons/sec) in indicated regions. (E) Temporal changes in mouse body weight at indicated dpi with initial body weight set to 100%. (F) Kaplan-Meier survival curves of mice statistically compared by log-rank (Mantel-Cox) test. for experiment as in A. (G) Viral loads (nLuc activity/g) in indicated organs using Vero E6 cells as targets. Undetectable virus amounts were set to 1. (H, I) Cytokine mRNA levels in lung and brain tissues after necropsy. The values were normalized to Gapdh in the same sample and that in uninfected mice. Viral loads (G) and inflammatory cytokine profile (H, I) was determined after necropsy at times indicated in B. Each curve in (C)-(E) and each data point in (G)-(I) represents an individual mouse. CV3-1 treatment times are indicated in (C)-(E). Grouped data in (C)-(I) were analyzed by 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests. Statistical significance for group comparisons to isotype control are shown in black and between CV3-1 and CV3-1 LALA treated cohorts are shown in red. ∗, p
    Figure Legend Snippet: Fc-mediated Antibody Effector Functions Contribute to the In Vivo Efficacy of CV3-1 (A) Experimental design to test therapeutic efficacy of NAb CV3-1 and its corresponding Leucine to Alanine (LALA) mutant administered ip (12.5 mg/kg body weight) 3 dpi to K18-hACE2 mice challenged with SARS-CoV-2 nLuc followed by non-invasive BLI every 2 days. Human IgG1-treated (12.5 mg/kg body weight) mice were used as the control cohort. (B) Representative images from temporal BLI of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions at the indicated dpi and after necropsy. Scale bars denote radiance (photons/sec/cm 2 /steradian). (C-D) Temporal quantification of nLuc signal acquired non-invasively as flux (photons/sec) in indicated regions. (E) Temporal changes in mouse body weight at indicated dpi with initial body weight set to 100%. (F) Kaplan-Meier survival curves of mice statistically compared by log-rank (Mantel-Cox) test. for experiment as in A. (G) Viral loads (nLuc activity/g) in indicated organs using Vero E6 cells as targets. Undetectable virus amounts were set to 1. (H, I) Cytokine mRNA levels in lung and brain tissues after necropsy. The values were normalized to Gapdh in the same sample and that in uninfected mice. Viral loads (G) and inflammatory cytokine profile (H, I) was determined after necropsy at times indicated in B. Each curve in (C)-(E) and each data point in (G)-(I) represents an individual mouse. CV3-1 treatment times are indicated in (C)-(E). Grouped data in (C)-(I) were analyzed by 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests. Statistical significance for group comparisons to isotype control are shown in black and between CV3-1 and CV3-1 LALA treated cohorts are shown in red. ∗, p

    Techniques Used: In Vivo, Mutagenesis, Mouse Assay, Infection, Activity Assay

    9) Product Images from "N-Glycans on the Rift Valley Fever Virus Envelope Glycoproteins Gn and Gc Redundantly Support Viral Infection via DC-SIGN"

    Article Title: N-Glycans on the Rift Valley Fever Virus Envelope Glycoproteins Gn and Gc Redundantly Support Viral Infection via DC-SIGN

    Journal: Viruses

    doi: 10.3390/v8050149

    Generation of recombinant MP-12 encoding asparagine (N)-to-glutamine (Q) mutation(s) in Gn/Gc sequons. The Gn/Gc migration patterns of rMP-12, or that encoding N-to-Q mutation either in Gn or Gc (N438Q, N794Q, N1035Q, and N1077Q mutants) ( A ) or that encoding N-to-Q mutations both in Gn and Gc (N438Q/N794Q, N438Q/N1035Q, and N438Q/N1077Q mutants) ( B ). Vero E6 cells were infected with rMP-12 or the mutants at a multiplicity of infection (MOI) of 0.1 to 3, and metabolically labeled with [ 35 S] methionine/cysteine from 1 to 16 hours post infection (hpi). The cleared culture supernatants were subjected to immunoprecipitation using anti-Rift Valley fever virus (RVFV) antibody. Precipitated virions were analyzed by 7.5% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Gc-large: slow migrating Gc; Gc-small: fast migrating Gc; ( C ) Virus growth kinetics in Vero cells. Vero cells were infected with indicated rMP-12 mutants at an MOI of 0.01. Virus titers were determined at 1, 24, 48, 72 and 96 hpi. Means +/− standard deviations of three independent experiments are shown.
    Figure Legend Snippet: Generation of recombinant MP-12 encoding asparagine (N)-to-glutamine (Q) mutation(s) in Gn/Gc sequons. The Gn/Gc migration patterns of rMP-12, or that encoding N-to-Q mutation either in Gn or Gc (N438Q, N794Q, N1035Q, and N1077Q mutants) ( A ) or that encoding N-to-Q mutations both in Gn and Gc (N438Q/N794Q, N438Q/N1035Q, and N438Q/N1077Q mutants) ( B ). Vero E6 cells were infected with rMP-12 or the mutants at a multiplicity of infection (MOI) of 0.1 to 3, and metabolically labeled with [ 35 S] methionine/cysteine from 1 to 16 hours post infection (hpi). The cleared culture supernatants were subjected to immunoprecipitation using anti-Rift Valley fever virus (RVFV) antibody. Precipitated virions were analyzed by 7.5% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Gc-large: slow migrating Gc; Gc-small: fast migrating Gc; ( C ) Virus growth kinetics in Vero cells. Vero cells were infected with indicated rMP-12 mutants at an MOI of 0.01. Virus titers were determined at 1, 24, 48, 72 and 96 hpi. Means +/− standard deviations of three independent experiments are shown.

    Techniques Used: Recombinant, Mutagenesis, Migration, Infection, Metabolic Labelling, Labeling, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Autoradiography

    10) Product Images from "Isolation, Sequence, Infectivity, and Replication Kinetics of Severe Acute Respiratory Syndrome Coronavirus 2"

    Article Title: Isolation, Sequence, Infectivity, and Replication Kinetics of Severe Acute Respiratory Syndrome Coronavirus 2

    Journal: Emerging Infectious Diseases

    doi: 10.3201/eid2609.201495

    Isolating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from patients with coronavirus disease (COVID-19). A) Vero E6 cells were mock inoculated or inoculated with midturbinate clinical specimens from COVID-19 patients. Cells were incubated for 72 h and observed for cytopathic effect (CPE) under a light microscope. Original magnification ×10. B) To determine if supernatant from Vero E6 cells that were mock inoculated or inoculated with clinical specimens contained replication competent virus, we reinoculated a fresh monolayer of Vero E6 cells and observed cells under a light microscope for CPE after 24 h. Original magnification ×10. C) Quantitative real-time PCR was used to detect SARS-CoV-2 5′-UTR and E gene in RNA extracted from supernatant that was collected from Vero E6 cells that were mock infected or infected with clinical specimens from COVID-19 patients for 72 h. D) Electron micrograph of Vero E6 cells that were reinfected for 48 h with supernatant that was collected from Vero E6 cells infected with clinical specimens. Original magnification ×36,000. Inset, zoomed and cropped from the original electron micrograph, shows coronavirus-like particles. M, mock specimen; specimen 1, SARS-CoV-2/SB2; specimen 2, SARS-CoV-2/SB3-TYAGNC. E, envelope; UTR, untranslated region.
    Figure Legend Snippet: Isolating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from patients with coronavirus disease (COVID-19). A) Vero E6 cells were mock inoculated or inoculated with midturbinate clinical specimens from COVID-19 patients. Cells were incubated for 72 h and observed for cytopathic effect (CPE) under a light microscope. Original magnification ×10. B) To determine if supernatant from Vero E6 cells that were mock inoculated or inoculated with clinical specimens contained replication competent virus, we reinoculated a fresh monolayer of Vero E6 cells and observed cells under a light microscope for CPE after 24 h. Original magnification ×10. C) Quantitative real-time PCR was used to detect SARS-CoV-2 5′-UTR and E gene in RNA extracted from supernatant that was collected from Vero E6 cells that were mock infected or infected with clinical specimens from COVID-19 patients for 72 h. D) Electron micrograph of Vero E6 cells that were reinfected for 48 h with supernatant that was collected from Vero E6 cells infected with clinical specimens. Original magnification ×36,000. Inset, zoomed and cropped from the original electron micrograph, shows coronavirus-like particles. M, mock specimen; specimen 1, SARS-CoV-2/SB2; specimen 2, SARS-CoV-2/SB3-TYAGNC. E, envelope; UTR, untranslated region.

    Techniques Used: Incubation, Light Microscopy, Real-time Polymerase Chain Reaction, Infection

    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein detection in infected Vero E6 and CD4+ T cells. To detect SARS-CoV-2 protein expression, we infected Vero E6 and CD4+ T cells with SARS-CoV-2 at a multiplicity of infection of 0.1 for 24 h. We immunostained these cells and observed them by using fluorescent microscopy. A) SARS-CoV-2–infected and immunostained Vero E6 cells. B) SARS-CoV-2–infected and immunostained CD4+ T cells. For panels A and B, cells were stained by using an antibody cocktail consisting of SARS-CoV-2 S1 antibody, SARS-CoV-2 N antibody, and diluted serum from a recovered coronavirus disease patient. C) SARS-CoV-2 infected CD4+ T cells immunostained with SARS-CoV-2 S1 antibody (anti-S). Scale bars indicate 400 μm; original magnification ×10.
    Figure Legend Snippet: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein detection in infected Vero E6 and CD4+ T cells. To detect SARS-CoV-2 protein expression, we infected Vero E6 and CD4+ T cells with SARS-CoV-2 at a multiplicity of infection of 0.1 for 24 h. We immunostained these cells and observed them by using fluorescent microscopy. A) SARS-CoV-2–infected and immunostained Vero E6 cells. B) SARS-CoV-2–infected and immunostained CD4+ T cells. For panels A and B, cells were stained by using an antibody cocktail consisting of SARS-CoV-2 S1 antibody, SARS-CoV-2 N antibody, and diluted serum from a recovered coronavirus disease patient. C) SARS-CoV-2 infected CD4+ T cells immunostained with SARS-CoV-2 S1 antibody (anti-S). Scale bars indicate 400 μm; original magnification ×10.

    Techniques Used: Infection, Expressing, Microscopy, Staining

    Replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human structural and immune cells. To identify human cells that support SARS-CoV-2 replication, we infected human cell lines and primary cells at a multiplicity of infection of 0.01 (n = 2 independent experiments; supernatant from each experiment was titrated in triplicate). We infected Vero E6 cells as a control. THF (human telomerase life-extended cells) and Calu-3 cells (human lung adenocarcinoma–derived) cells represent human structural cells. THP-1 is a monocyte cell line that was used to derive macrophages and dendritic cells. PBMCs from 2 healthy human donors were used to generate CD4+, CD8+, CD19+, monocytes, and other (CD4–, CD8–, CD19–) cell populations. Supernatant from infected cells was collected at various times and titrated on Vero E6 cells to determine virus titers (TCID 50 ). PBMC, peripheral blood mononuclear cell; TCID 50 , 50% tissue culture infectious dose.
    Figure Legend Snippet: Replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human structural and immune cells. To identify human cells that support SARS-CoV-2 replication, we infected human cell lines and primary cells at a multiplicity of infection of 0.01 (n = 2 independent experiments; supernatant from each experiment was titrated in triplicate). We infected Vero E6 cells as a control. THF (human telomerase life-extended cells) and Calu-3 cells (human lung adenocarcinoma–derived) cells represent human structural cells. THP-1 is a monocyte cell line that was used to derive macrophages and dendritic cells. PBMCs from 2 healthy human donors were used to generate CD4+, CD8+, CD19+, monocytes, and other (CD4–, CD8–, CD19–) cell populations. Supernatant from infected cells was collected at various times and titrated on Vero E6 cells to determine virus titers (TCID 50 ). PBMC, peripheral blood mononuclear cell; TCID 50 , 50% tissue culture infectious dose.

    Techniques Used: Infection, Derivative Assay

    Electron micrographs of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–infected cells. To detect coronavirus-like particles in experimentally infected human structural and immune cells, we infected a range of cells with SARS-CoV-2 at a multiplicity of infection of 0.01 for 48 h. The cells were fixed, processed, and imaged by using a transmission electron microscope (10 fields/cell type). A representative image of each cell type is shown. Virus-like particles are indicated by red arrows. A) Vero E6 cells. B) Calu-3 cells. C) CD4+ PBMCs. D) CD8+ PBMCs. E) CD19+ PBMCs. F) Monocytes from PBMCs. G) Other cells from PBMCs (CD4–, CD8–, CD19– cell populations). H) THP-1 monocyte. I) THP-1-derived macrophage. J) THP-1-derived dendritic cell. PBMC, peripheral blood mononuclear cell. Scale bars indicate 200 nm.
    Figure Legend Snippet: Electron micrographs of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–infected cells. To detect coronavirus-like particles in experimentally infected human structural and immune cells, we infected a range of cells with SARS-CoV-2 at a multiplicity of infection of 0.01 for 48 h. The cells were fixed, processed, and imaged by using a transmission electron microscope (10 fields/cell type). A representative image of each cell type is shown. Virus-like particles are indicated by red arrows. A) Vero E6 cells. B) Calu-3 cells. C) CD4+ PBMCs. D) CD8+ PBMCs. E) CD19+ PBMCs. F) Monocytes from PBMCs. G) Other cells from PBMCs (CD4–, CD8–, CD19– cell populations). H) THP-1 monocyte. I) THP-1-derived macrophage. J) THP-1-derived dendritic cell. PBMC, peripheral blood mononuclear cell. Scale bars indicate 200 nm.

    Techniques Used: Infection, Transmission Assay, Microscopy, Derivative Assay

    11) Product Images from "Sangassou Virus, the First Hantavirus Isolate from Africa, Displays Genetic and Functional Properties Distinct from Those of Other Murinae-Associated Hantaviruses"

    Article Title: Sangassou Virus, the First Hantavirus Isolate from Africa, Displays Genetic and Functional Properties Distinct from Those of Other Murinae-Associated Hantaviruses

    Journal: Journal of Virology

    doi: 10.1128/JVI.05879-11

    Inhibition of SANGV infection in vitro by application of specific monoclonal antibodies against β 1 integrin, β 3 integrin, and decay-accelerating factor (DAF) in Vero E6 cells. Efficiency of inhibition was monitored in comparison to HTNV
    Figure Legend Snippet: Inhibition of SANGV infection in vitro by application of specific monoclonal antibodies against β 1 integrin, β 3 integrin, and decay-accelerating factor (DAF) in Vero E6 cells. Efficiency of inhibition was monitored in comparison to HTNV

    Techniques Used: Inhibition, Infection, In Vitro

    Sangassou virus induces IFN-λ expression in IFN type I-deficient Vero E6 cells. IFN-λ is responsible for early activation of the MxA gene in infected A549 cells. (A) Concentration of IFN-λ1 present in virus stocks measured by ELISA.
    Figure Legend Snippet: Sangassou virus induces IFN-λ expression in IFN type I-deficient Vero E6 cells. IFN-λ is responsible for early activation of the MxA gene in infected A549 cells. (A) Concentration of IFN-λ1 present in virus stocks measured by ELISA.

    Techniques Used: Expressing, Activation Assay, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay

    12) Product Images from "Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis"

    Article Title: Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

    Journal: Journal of Virology

    doi: 10.1128/JVI.03491-13

    siRNAs targeting α5 and αv back cell-cell fusion triggered by hMPV F protein. (A) Knockdown of integrin α5 and αv expression by siRNA. Twenty picomoles of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA was transfected into Vero E6 cells in 24-well plates using Oligofectamine reagents according to the manufacturer's instructions. After 48 h posttransfection, the expression of α5 or αv was detected by Western blotting. (B) Syncytium formation induced by F protein of hMPV after knockdown of integrins α5 and αv. Vero E6 cells in 24-well plates were transfected with 20 pmol of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA. After treatment with siRNAs for 24 h, Vero E6 cells were transfected with 0.8 μg of pCAGGS-F using Lipofectamine Plus reagents and then subjected to pH pulses (pH 5.0). At 48 h, monolayers were fixed with methanol and stained with Giemsa. (C) Quantitation of syncytium formation after knockdown of integrins α5 and αv. The number of syncytia (≥4 nuclei in each syncytium) was counted under a microscope using six randomly selected fields in each siRNA-treated or untreated well. The mean number of syncytia per field was calculated for each treatment. The percent fusion for each siRNA treatment was normalized by the mean number of syncytia in cells transfected with pCAGGS-F without siRNA treatment. The data shown are averages for three independent experiments.
    Figure Legend Snippet: siRNAs targeting α5 and αv back cell-cell fusion triggered by hMPV F protein. (A) Knockdown of integrin α5 and αv expression by siRNA. Twenty picomoles of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA was transfected into Vero E6 cells in 24-well plates using Oligofectamine reagents according to the manufacturer's instructions. After 48 h posttransfection, the expression of α5 or αv was detected by Western blotting. (B) Syncytium formation induced by F protein of hMPV after knockdown of integrins α5 and αv. Vero E6 cells in 24-well plates were transfected with 20 pmol of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA. After treatment with siRNAs for 24 h, Vero E6 cells were transfected with 0.8 μg of pCAGGS-F using Lipofectamine Plus reagents and then subjected to pH pulses (pH 5.0). At 48 h, monolayers were fixed with methanol and stained with Giemsa. (C) Quantitation of syncytium formation after knockdown of integrins α5 and αv. The number of syncytia (≥4 nuclei in each syncytium) was counted under a microscope using six randomly selected fields in each siRNA-treated or untreated well. The mean number of syncytia per field was calculated for each treatment. The percent fusion for each siRNA treatment was normalized by the mean number of syncytia in cells transfected with pCAGGS-F without siRNA treatment. The data shown are averages for three independent experiments.

    Techniques Used: Expressing, Transfection, Western Blot, Staining, Quantitation Assay, Microscopy

    Effects of mutations to the RGD motif on cell-cell fusion triggered by hMPV F protein. (A) Syncytium formation of hMPV F proteins carrying mutations at the RGD motif. Confluent monolayers of Vero E6 cells were transfected with 2 μg plasmids of pCAGGS-F or F-protein mutants. At 24 h posttransfection, the monolayers were fixed with methanol and stained with Giemsa. (B) Content-mixing fusion assay for hMPV F mutants. The extent of fusion for each F mutant was quantitated with the content-mixing fusion assay at pH 5.0 and normalized by the fusion of wild-type hMPV F protein. The data shown are averages for three independent experiments. (C) Cell surface expression of hMPV F mutants. Cell surface expression was determined by FACS using monoclonal antibody against hMPV F protein and normalized by the expression level of wild-type F protein at the cell surface.
    Figure Legend Snippet: Effects of mutations to the RGD motif on cell-cell fusion triggered by hMPV F protein. (A) Syncytium formation of hMPV F proteins carrying mutations at the RGD motif. Confluent monolayers of Vero E6 cells were transfected with 2 μg plasmids of pCAGGS-F or F-protein mutants. At 24 h posttransfection, the monolayers were fixed with methanol and stained with Giemsa. (B) Content-mixing fusion assay for hMPV F mutants. The extent of fusion for each F mutant was quantitated with the content-mixing fusion assay at pH 5.0 and normalized by the fusion of wild-type hMPV F protein. The data shown are averages for three independent experiments. (C) Cell surface expression of hMPV F mutants. Cell surface expression was determined by FACS using monoclonal antibody against hMPV F protein and normalized by the expression level of wild-type F protein at the cell surface.

    Techniques Used: Transfection, Staining, Single Vesicle Fusion Assay, Mutagenesis, Expressing, FACS

    Recovery of recombinant hMPVs carrying mutations in the RGD motif. (Top) Immunostaining spots formed by recombinant hMPVs. LLC-MK2 cells were infected with recombinant hMPV mutants and incubated at 37°C for 1 h. At day 4 postinfection, the supernatant was removed and cells were fixed. The cells were then labeled with an anti-hMPV N protein primary monoclonal antibody, followed by incubation with HRP-labeled rabbit antimouse secondary antibody. After incubation with AEC chromogen substrate, positive cells with immunostaining spots were visualized under a microscope. (Bottom) Plaque morphology of recombinant hMPVs. An agarose overlay plaque assay was performed in monolayer Vero E6 cells. Viral plaques were developed at day 7 postinfection.
    Figure Legend Snippet: Recovery of recombinant hMPVs carrying mutations in the RGD motif. (Top) Immunostaining spots formed by recombinant hMPVs. LLC-MK2 cells were infected with recombinant hMPV mutants and incubated at 37°C for 1 h. At day 4 postinfection, the supernatant was removed and cells were fixed. The cells were then labeled with an anti-hMPV N protein primary monoclonal antibody, followed by incubation with HRP-labeled rabbit antimouse secondary antibody. After incubation with AEC chromogen substrate, positive cells with immunostaining spots were visualized under a microscope. (Bottom) Plaque morphology of recombinant hMPVs. An agarose overlay plaque assay was performed in monolayer Vero E6 cells. Viral plaques were developed at day 7 postinfection.

    Techniques Used: Recombinant, Immunostaining, Infection, Incubation, Labeling, Microscopy, Plaque Assay

    13) Product Images from "Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis"

    Article Title: Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

    Journal: Journal of Virology

    doi: 10.1128/JVI.03491-13

    siRNAs targeting α5 and αv back cell-cell fusion triggered by hMPV F protein. (A) Knockdown of integrin α5 and αv expression by siRNA. Twenty picomoles of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA was transfected into Vero E6 cells in 24-well plates using Oligofectamine reagents according to the manufacturer's instructions. After 48 h posttransfection, the expression of α5 or αv was detected by Western blotting. (B) Syncytium formation induced by F protein of hMPV after knockdown of integrins α5 and αv. Vero E6 cells in 24-well plates were transfected with 20 pmol of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA. After treatment with siRNAs for 24 h, Vero E6 cells were transfected with 0.8 μg of pCAGGS-F using Lipofectamine Plus reagents and then subjected to pH pulses (pH 5.0). At 48 h, monolayers were fixed with methanol and stained with Giemsa. (C) Quantitation of syncytium formation after knockdown of integrins α5 and αv. The number of syncytia (≥4 nuclei in each syncytium) was counted under a microscope using six randomly selected fields in each siRNA-treated or untreated well. The mean number of syncytia per field was calculated for each treatment. The percent fusion for each siRNA treatment was normalized by the mean number of syncytia in cells transfected with pCAGGS-F without siRNA treatment. The data shown are averages for three independent experiments.
    Figure Legend Snippet: siRNAs targeting α5 and αv back cell-cell fusion triggered by hMPV F protein. (A) Knockdown of integrin α5 and αv expression by siRNA. Twenty picomoles of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA was transfected into Vero E6 cells in 24-well plates using Oligofectamine reagents according to the manufacturer's instructions. After 48 h posttransfection, the expression of α5 or αv was detected by Western blotting. (B) Syncytium formation induced by F protein of hMPV after knockdown of integrins α5 and αv. Vero E6 cells in 24-well plates were transfected with 20 pmol of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA. After treatment with siRNAs for 24 h, Vero E6 cells were transfected with 0.8 μg of pCAGGS-F using Lipofectamine Plus reagents and then subjected to pH pulses (pH 5.0). At 48 h, monolayers were fixed with methanol and stained with Giemsa. (C) Quantitation of syncytium formation after knockdown of integrins α5 and αv. The number of syncytia (≥4 nuclei in each syncytium) was counted under a microscope using six randomly selected fields in each siRNA-treated or untreated well. The mean number of syncytia per field was calculated for each treatment. The percent fusion for each siRNA treatment was normalized by the mean number of syncytia in cells transfected with pCAGGS-F without siRNA treatment. The data shown are averages for three independent experiments.

    Techniques Used: Expressing, Transfection, Western Blot, Staining, Quantitation Assay, Microscopy

    Effects of mutations to the RGD motif on cell-cell fusion triggered by hMPV F protein. (A) Syncytium formation of hMPV F proteins carrying mutations at the RGD motif. Confluent monolayers of Vero E6 cells were transfected with 2 μg plasmids of pCAGGS-F or F-protein mutants. At 24 h posttransfection, the monolayers were fixed with methanol and stained with Giemsa. (B) Content-mixing fusion assay for hMPV F mutants. The extent of fusion for each F mutant was quantitated with the content-mixing fusion assay at pH 5.0 and normalized by the fusion of wild-type hMPV F protein. The data shown are averages for three independent experiments. (C) Cell surface expression of hMPV F mutants. Cell surface expression was determined by FACS using monoclonal antibody against hMPV F protein and normalized by the expression level of wild-type F protein at the cell surface.
    Figure Legend Snippet: Effects of mutations to the RGD motif on cell-cell fusion triggered by hMPV F protein. (A) Syncytium formation of hMPV F proteins carrying mutations at the RGD motif. Confluent monolayers of Vero E6 cells were transfected with 2 μg plasmids of pCAGGS-F or F-protein mutants. At 24 h posttransfection, the monolayers were fixed with methanol and stained with Giemsa. (B) Content-mixing fusion assay for hMPV F mutants. The extent of fusion for each F mutant was quantitated with the content-mixing fusion assay at pH 5.0 and normalized by the fusion of wild-type hMPV F protein. The data shown are averages for three independent experiments. (C) Cell surface expression of hMPV F mutants. Cell surface expression was determined by FACS using monoclonal antibody against hMPV F protein and normalized by the expression level of wild-type F protein at the cell surface.

    Techniques Used: Transfection, Staining, Single Vesicle Fusion Assay, Mutagenesis, Expressing, FACS

    Recovery of recombinant hMPVs carrying mutations in the RGD motif. (Top) Immunostaining spots formed by recombinant hMPVs. LLC-MK2 cells were infected with recombinant hMPV mutants and incubated at 37°C for 1 h. At day 4 postinfection, the supernatant was removed and cells were fixed. The cells were then labeled with an anti-hMPV N protein primary monoclonal antibody, followed by incubation with HRP-labeled rabbit antimouse secondary antibody. After incubation with AEC chromogen substrate, positive cells with immunostaining spots were visualized under a microscope. (Bottom) Plaque morphology of recombinant hMPVs. An agarose overlay plaque assay was performed in monolayer Vero E6 cells. Viral plaques were developed at day 7 postinfection.
    Figure Legend Snippet: Recovery of recombinant hMPVs carrying mutations in the RGD motif. (Top) Immunostaining spots formed by recombinant hMPVs. LLC-MK2 cells were infected with recombinant hMPV mutants and incubated at 37°C for 1 h. At day 4 postinfection, the supernatant was removed and cells were fixed. The cells were then labeled with an anti-hMPV N protein primary monoclonal antibody, followed by incubation with HRP-labeled rabbit antimouse secondary antibody. After incubation with AEC chromogen substrate, positive cells with immunostaining spots were visualized under a microscope. (Bottom) Plaque morphology of recombinant hMPVs. An agarose overlay plaque assay was performed in monolayer Vero E6 cells. Viral plaques were developed at day 7 postinfection.

    Techniques Used: Recombinant, Immunostaining, Infection, Incubation, Labeling, Microscopy, Plaque Assay

    14) Product Images from "A Small interfering RNA lead targeting RNA-dependent RNA-polymerase effectively inhibit the SARS-CoV-2 infection in Golden Syrian hamster and Rhesus macaque"

    Article Title: A Small interfering RNA lead targeting RNA-dependent RNA-polymerase effectively inhibit the SARS-CoV-2 infection in Golden Syrian hamster and Rhesus macaque

    Journal: bioRxiv

    doi: 10.1101/2020.07.07.190967

    siRNA No. 14 inhibition of SARS-CoV-2 cytopathicity in Vero E6 cells (CPE assay and EC 50 ). Vero E6 cells were infected with SARS-CoV-2 and incubated for 2 days. (a) Mock-siRNA (100 nM). (b) 5 nM. (c) 10 nM. (d) 20 nM. (e) 30 nM. (f) 40 nM. (g) 50 nM. (h) 60 nM. (i) 70 nM. (j) 80 nM. (k) 90 nM. (l) 100 nM. (m) EC 50 of siRNA No. 14 using qRT-PCR.
    Figure Legend Snippet: siRNA No. 14 inhibition of SARS-CoV-2 cytopathicity in Vero E6 cells (CPE assay and EC 50 ). Vero E6 cells were infected with SARS-CoV-2 and incubated for 2 days. (a) Mock-siRNA (100 nM). (b) 5 nM. (c) 10 nM. (d) 20 nM. (e) 30 nM. (f) 40 nM. (g) 50 nM. (h) 60 nM. (i) 70 nM. (j) 80 nM. (k) 90 nM. (l) 100 nM. (m) EC 50 of siRNA No. 14 using qRT-PCR.

    Techniques Used: Inhibition, Infection, Incubation, Quantitative RT-PCR

    15) Product Images from "Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell"

    Article Title: Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell

    Journal: bioRxiv

    doi: 10.1101/2021.03.01.433431

    The H522 cell line is null for ACE2 expression and is permissive to SARS-CoV-2 infection. A, Normalized RNA-seq reads were aligned to the GRCh38 and Vervet-African green monkey genomes and quantified with Salmon (v1.3.0). The read counts for ACE2, TMPRSS2, FURIN, CTSB, CTSL , and NRP1 are given for the indicated cell lines. See also Figure S1 and Table S1. B, qRT-PCR for ACE2 and TMPRSS2 expression normalized to 1μg input RNA for each cell line. Cercopithecus aethiops specific primers against TMPRSS2 were used for the Vero E6 samples. Each bar represents mean, error bars indicate SEM (n=3). C, Immunoblot showing ACE2 expression across 10 lung and upper airway cancer cell lines and Vero E6 cells (representative of n=3). ACE2 expression was quantified using Licor Image Studio software in which ACE2 levels were normalized to β-ACTIN, set relative to Vero E6, and are indicated below the immunoblots. D, qRT-PCR for cell-associated SARS-CoV-2 RNA at 4 and 72 hpi at MOI=0.015 or 0.15. MOIs were determined by titration on Vero E6 cells. Error bars represent SEM (n=3). * indicates p
    Figure Legend Snippet: The H522 cell line is null for ACE2 expression and is permissive to SARS-CoV-2 infection. A, Normalized RNA-seq reads were aligned to the GRCh38 and Vervet-African green monkey genomes and quantified with Salmon (v1.3.0). The read counts for ACE2, TMPRSS2, FURIN, CTSB, CTSL , and NRP1 are given for the indicated cell lines. See also Figure S1 and Table S1. B, qRT-PCR for ACE2 and TMPRSS2 expression normalized to 1μg input RNA for each cell line. Cercopithecus aethiops specific primers against TMPRSS2 were used for the Vero E6 samples. Each bar represents mean, error bars indicate SEM (n=3). C, Immunoblot showing ACE2 expression across 10 lung and upper airway cancer cell lines and Vero E6 cells (representative of n=3). ACE2 expression was quantified using Licor Image Studio software in which ACE2 levels were normalized to β-ACTIN, set relative to Vero E6, and are indicated below the immunoblots. D, qRT-PCR for cell-associated SARS-CoV-2 RNA at 4 and 72 hpi at MOI=0.015 or 0.15. MOIs were determined by titration on Vero E6 cells. Error bars represent SEM (n=3). * indicates p

    Techniques Used: Expressing, Infection, RNA Sequencing Assay, Quantitative RT-PCR, Software, Western Blot, Titration

    16) Product Images from "An artificial intelligence system reveals liquiritin inhibits SARS-CoV-2 by mimicking type I interferon"

    Article Title: An artificial intelligence system reveals liquiritin inhibits SARS-CoV-2 by mimicking type I interferon

    Journal: bioRxiv

    doi: 10.1101/2020.05.02.074021

    The antiviral activities of the liquiritin against SARS-CoV-2 in vitro . a) The workflow of the computation of enrichment score (ES). b) The ES score of all compounds. Liquiritin ranks first. c) The antiviral activity of liquiritin in Vero E6 cells (EC 50 = 2.39 μM, n = 3 for where error bar is shown). d) Cytotoxicity of the liquiritin to Vero E6 cells was measured by MTS assay.
    Figure Legend Snippet: The antiviral activities of the liquiritin against SARS-CoV-2 in vitro . a) The workflow of the computation of enrichment score (ES). b) The ES score of all compounds. Liquiritin ranks first. c) The antiviral activity of liquiritin in Vero E6 cells (EC 50 = 2.39 μM, n = 3 for where error bar is shown). d) Cytotoxicity of the liquiritin to Vero E6 cells was measured by MTS assay.

    Techniques Used: In Vitro, Activity Assay, MTS Assay

    17) Product Images from "Neutralizing Monoclonal Antibodies against the Gn and the Gc of the Andes Virus Glycoprotein Spike Complex Protect from Virus Challenge in a Preclinical Hamster Model"

    Article Title: Neutralizing Monoclonal Antibodies against the Gn and the Gc of the Andes Virus Glycoprotein Spike Complex Protect from Virus Challenge in a Preclinical Hamster Model

    Journal: mBio

    doi: 10.1128/mBio.00028-20

    Neutralization and effector functions of the isolated MAbs. (A) FRNAs of “AN” fusion MAbs against VSV-ANDV. (B) FRNAs of “HAP” fusion MAbs against VSV-ANDV. For both A and B, each MAb was run in duplicates in 3-fold serial dilutions starting at 30 μg/ml. Any MAb exhibiting > 15% neutralization at the last concentration was repeated with lower dilutions. The negative-control MAb was a murine IgG2a specific for Zaire ebolavirus (KL-2G12). All trend lines are logarithmic regressions except where such regressions were not converged. (C) FRNAs conducted as for panels A and B but against authentic ANDV. Dashed lines in panels A, B, and C demonstrate 50% inhibition. (D) Comparison of IC 50 values of each MAb against VSV-ANDV and authentic ANDV. Error bars indicate 95% confidence intervals. (E and F) ADCC reporter assays of each MAb against VSV-ANDV-infected Vero.E6 cells (MOI, 1.0). Data shown are the result from one experiment with a shared positive control in panels E and F (serum from homologous fusion). This positive control was used in 3-fold serial dilutions with a starting dilution of 1:300.
    Figure Legend Snippet: Neutralization and effector functions of the isolated MAbs. (A) FRNAs of “AN” fusion MAbs against VSV-ANDV. (B) FRNAs of “HAP” fusion MAbs against VSV-ANDV. For both A and B, each MAb was run in duplicates in 3-fold serial dilutions starting at 30 μg/ml. Any MAb exhibiting > 15% neutralization at the last concentration was repeated with lower dilutions. The negative-control MAb was a murine IgG2a specific for Zaire ebolavirus (KL-2G12). All trend lines are logarithmic regressions except where such regressions were not converged. (C) FRNAs conducted as for panels A and B but against authentic ANDV. Dashed lines in panels A, B, and C demonstrate 50% inhibition. (D) Comparison of IC 50 values of each MAb against VSV-ANDV and authentic ANDV. Error bars indicate 95% confidence intervals. (E and F) ADCC reporter assays of each MAb against VSV-ANDV-infected Vero.E6 cells (MOI, 1.0). Data shown are the result from one experiment with a shared positive control in panels E and F (serum from homologous fusion). This positive control was used in 3-fold serial dilutions with a starting dilution of 1:300.

    Techniques Used: Neutralization, Isolation, Concentration Assay, Negative Control, Inhibition, Infection, Positive Control

    18) Product Images from "Monoclonal Antibodies with Neutralizing Activity and Fc-Effector Functions against the Machupo Virus Glycoprotein"

    Article Title: Monoclonal Antibodies with Neutralizing Activity and Fc-Effector Functions against the Machupo Virus Glycoprotein

    Journal: Journal of Virology

    doi: 10.1128/JVI.01741-19

    Antibodies bind to cells infected with VSV-MACV. (A) Binding of antibodies to MACV glycoprotein in an immunofluorescence assay. Vero.E6 cells were infected with VSV-MACV at an MOI of 1.0 overnight and then fixed and immunostained using each antibody at a concentration of 30 μg/ml. An irrelevant antibody, anti-influenza H4 virus (KL-H4-4A11), was used as a negative control while the positive control used was KL-AV-2A1. (B) Western blot analysis. A standard Western blot was performed using recombinant MACV GPC and an irrelevant protein, influenza virus H2 HA. Each blot was stained with 30 μg/ml of each respective antibody, and an anti-mouse IgG conjugated to alkaline phosphatase was used as a secondary antibody at a dilution of 1:3,000. A separate Western blot was run using anti-histidine antibody to ensure proper transfer of protein to the nitrocellulose membrane. The ladder used was a ColorPlus Prestained Protein Ladder, Broad Range (New England BioLabs).
    Figure Legend Snippet: Antibodies bind to cells infected with VSV-MACV. (A) Binding of antibodies to MACV glycoprotein in an immunofluorescence assay. Vero.E6 cells were infected with VSV-MACV at an MOI of 1.0 overnight and then fixed and immunostained using each antibody at a concentration of 30 μg/ml. An irrelevant antibody, anti-influenza H4 virus (KL-H4-4A11), was used as a negative control while the positive control used was KL-AV-2A1. (B) Western blot analysis. A standard Western blot was performed using recombinant MACV GPC and an irrelevant protein, influenza virus H2 HA. Each blot was stained with 30 μg/ml of each respective antibody, and an anti-mouse IgG conjugated to alkaline phosphatase was used as a secondary antibody at a dilution of 1:3,000. A separate Western blot was run using anti-histidine antibody to ensure proper transfer of protein to the nitrocellulose membrane. The ladder used was a ColorPlus Prestained Protein Ladder, Broad Range (New England BioLabs).

    Techniques Used: Infection, Binding Assay, Immunofluorescence, Concentration Assay, Negative Control, Positive Control, Western Blot, Recombinant, Gel Permeation Chromatography, Staining

    19) Product Images from "The Human Sodium Iodide Symporter as a Reporter Gene for Studying Middle East Respiratory Syndrome Coronavirus Pathogenesis"

    Article Title: The Human Sodium Iodide Symporter as a Reporter Gene for Studying Middle East Respiratory Syndrome Coronavirus Pathogenesis

    Journal: mSphere

    doi: 10.1128/mSphere.00540-18

    Radio-uptake of 99m Tc-pertechnetate by planar scintigraphy. (a) Experimental overview of in vitro evaluation of the rMERS-CoV/ hNIS virus. Vero E6 cells were infected with rMERS-CoV or rMERS-CoV/ hNIS at an MOI of 0.01 or 0.04. At various time points postinfection, the cells were incubated with 99m Tc-pertechnetate, and images of the plates were acquired. (b) Plate layout for hNIS functional assays. (c) Representative images of the plates acquired at 24 h postinfection at an MOI of 0.01 (top plates) or 0.04 (bottom plates) after incubation with 99m Tc-pertechnetate.
    Figure Legend Snippet: Radio-uptake of 99m Tc-pertechnetate by planar scintigraphy. (a) Experimental overview of in vitro evaluation of the rMERS-CoV/ hNIS virus. Vero E6 cells were infected with rMERS-CoV or rMERS-CoV/ hNIS at an MOI of 0.01 or 0.04. At various time points postinfection, the cells were incubated with 99m Tc-pertechnetate, and images of the plates were acquired. (b) Plate layout for hNIS functional assays. (c) Representative images of the plates acquired at 24 h postinfection at an MOI of 0.01 (top plates) or 0.04 (bottom plates) after incubation with 99m Tc-pertechnetate.

    Techniques Used: In Vitro, Infection, Incubation, Functional Assay

    Retention of hNIS transgene following viral kinetics analysis and serial passage. (a and b) Vero E6 cells were infected with rMERS-CoV/hNIS (a) or parental rMERS-CoV (b) at an MOI of 0.01 or 3 and then collected at 96 h postinfection for RT-PCR. (c) Retention of the hNIS gene following serial passage. RNA was extracted from cells 72 h postinfection followed by RT-PCR at passage 6. A positive-control virus (C+) and uninfected negative-control cells (C−) were used as controls.
    Figure Legend Snippet: Retention of hNIS transgene following viral kinetics analysis and serial passage. (a and b) Vero E6 cells were infected with rMERS-CoV/hNIS (a) or parental rMERS-CoV (b) at an MOI of 0.01 or 3 and then collected at 96 h postinfection for RT-PCR. (c) Retention of the hNIS gene following serial passage. RNA was extracted from cells 72 h postinfection followed by RT-PCR at passage 6. A positive-control virus (C+) and uninfected negative-control cells (C−) were used as controls.

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control

    Kinetics of rMERS-CoV/ hNIS and parental rMERS-CoV replication in Vero E6 cells. (a and b) Multistep (a) and one-step (b) growth curves of Vero E6 cells infected with rMERS-CoV (Parental) and rMERS-CoV/ hNIS ( hNIS ). Quantification of the release of infectious virus at the indicated time points (hours postexposure) was determined by plaque assays. Each data point represents the mean ± standard deviation (SD) (error bar) averaged from three independent experiments. (c and d) Cytopathology of rMERS-CoV and rMERS-CoV/ hNIS in Vero E6 cells. The cells were infected with either rMERS-CoV or rMERS-CoV/ hNIS at an MOI of 0.01 (c) or 3 (d) and analyzed by light microscopy at the indicated time points. Photomicrographs were taken using a 40× objective.
    Figure Legend Snippet: Kinetics of rMERS-CoV/ hNIS and parental rMERS-CoV replication in Vero E6 cells. (a and b) Multistep (a) and one-step (b) growth curves of Vero E6 cells infected with rMERS-CoV (Parental) and rMERS-CoV/ hNIS ( hNIS ). Quantification of the release of infectious virus at the indicated time points (hours postexposure) was determined by plaque assays. Each data point represents the mean ± standard deviation (SD) (error bar) averaged from three independent experiments. (c and d) Cytopathology of rMERS-CoV and rMERS-CoV/ hNIS in Vero E6 cells. The cells were infected with either rMERS-CoV or rMERS-CoV/ hNIS at an MOI of 0.01 (c) or 3 (d) and analyzed by light microscopy at the indicated time points. Photomicrographs were taken using a 40× objective.

    Techniques Used: Infection, Standard Deviation, Light Microscopy

    20) Product Images from "Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis"

    Article Title: Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

    Journal: Journal of Virology

    doi: 10.1128/JVI.03491-13

    siRNAs targeting α5 and αv back cell-cell fusion triggered by hMPV F protein. (A) Knockdown of integrin α5 and αv expression by siRNA. Twenty picomoles of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA was transfected into Vero E6 cells in 24-well plates using Oligofectamine reagents according to the manufacturer's instructions. After 48 h posttransfection, the expression of α5 or αv was detected by Western blotting. (B) Syncytium formation induced by F protein of hMPV after knockdown of integrins α5 and αv. Vero E6 cells in 24-well plates were transfected with 20 pmol of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA. After treatment with siRNAs for 24 h, Vero E6 cells were transfected with 0.8 μg of pCAGGS-F using Lipofectamine Plus reagents and then subjected to pH pulses (pH 5.0). At 48 h, monolayers were fixed with methanol and stained with Giemsa. (C) Quantitation of syncytium formation after knockdown of integrins α5 and αv. The number of syncytia (≥4 nuclei in each syncytium) was counted under a microscope using six randomly selected fields in each siRNA-treated or untreated well. The mean number of syncytia per field was calculated for each treatment. The percent fusion for each siRNA treatment was normalized by the mean number of syncytia in cells transfected with pCAGGS-F without siRNA treatment. The data shown are averages for three independent experiments.
    Figure Legend Snippet: siRNAs targeting α5 and αv back cell-cell fusion triggered by hMPV F protein. (A) Knockdown of integrin α5 and αv expression by siRNA. Twenty picomoles of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA was transfected into Vero E6 cells in 24-well plates using Oligofectamine reagents according to the manufacturer's instructions. After 48 h posttransfection, the expression of α5 or αv was detected by Western blotting. (B) Syncytium formation induced by F protein of hMPV after knockdown of integrins α5 and αv. Vero E6 cells in 24-well plates were transfected with 20 pmol of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA. After treatment with siRNAs for 24 h, Vero E6 cells were transfected with 0.8 μg of pCAGGS-F using Lipofectamine Plus reagents and then subjected to pH pulses (pH 5.0). At 48 h, monolayers were fixed with methanol and stained with Giemsa. (C) Quantitation of syncytium formation after knockdown of integrins α5 and αv. The number of syncytia (≥4 nuclei in each syncytium) was counted under a microscope using six randomly selected fields in each siRNA-treated or untreated well. The mean number of syncytia per field was calculated for each treatment. The percent fusion for each siRNA treatment was normalized by the mean number of syncytia in cells transfected with pCAGGS-F without siRNA treatment. The data shown are averages for three independent experiments.

    Techniques Used: Expressing, Transfection, Western Blot, Staining, Quantitation Assay, Microscopy

    Effects of mutations to the RGD motif on cell-cell fusion triggered by hMPV F protein. (A) Syncytium formation of hMPV F proteins carrying mutations at the RGD motif. Confluent monolayers of Vero E6 cells were transfected with 2 μg plasmids of pCAGGS-F or F-protein mutants. At 24 h posttransfection, the monolayers were fixed with methanol and stained with Giemsa. (B) Content-mixing fusion assay for hMPV F mutants. The extent of fusion for each F mutant was quantitated with the content-mixing fusion assay at pH 5.0 and normalized by the fusion of wild-type hMPV F protein. The data shown are averages for three independent experiments. (C) Cell surface expression of hMPV F mutants. Cell surface expression was determined by FACS using monoclonal antibody against hMPV F protein and normalized by the expression level of wild-type F protein at the cell surface.
    Figure Legend Snippet: Effects of mutations to the RGD motif on cell-cell fusion triggered by hMPV F protein. (A) Syncytium formation of hMPV F proteins carrying mutations at the RGD motif. Confluent monolayers of Vero E6 cells were transfected with 2 μg plasmids of pCAGGS-F or F-protein mutants. At 24 h posttransfection, the monolayers were fixed with methanol and stained with Giemsa. (B) Content-mixing fusion assay for hMPV F mutants. The extent of fusion for each F mutant was quantitated with the content-mixing fusion assay at pH 5.0 and normalized by the fusion of wild-type hMPV F protein. The data shown are averages for three independent experiments. (C) Cell surface expression of hMPV F mutants. Cell surface expression was determined by FACS using monoclonal antibody against hMPV F protein and normalized by the expression level of wild-type F protein at the cell surface.

    Techniques Used: Transfection, Staining, Single Vesicle Fusion Assay, Mutagenesis, Expressing, FACS

    Recovery of recombinant hMPVs carrying mutations in the RGD motif. (Top) Immunostaining spots formed by recombinant hMPVs. LLC-MK2 cells were infected with recombinant hMPV mutants and incubated at 37°C for 1 h. At day 4 postinfection, the supernatant was removed and cells were fixed. The cells were then labeled with an anti-hMPV N protein primary monoclonal antibody, followed by incubation with HRP-labeled rabbit antimouse secondary antibody. After incubation with AEC chromogen substrate, positive cells with immunostaining spots were visualized under a microscope. (Bottom) Plaque morphology of recombinant hMPVs. An agarose overlay plaque assay was performed in monolayer Vero E6 cells. Viral plaques were developed at day 7 postinfection.
    Figure Legend Snippet: Recovery of recombinant hMPVs carrying mutations in the RGD motif. (Top) Immunostaining spots formed by recombinant hMPVs. LLC-MK2 cells were infected with recombinant hMPV mutants and incubated at 37°C for 1 h. At day 4 postinfection, the supernatant was removed and cells were fixed. The cells were then labeled with an anti-hMPV N protein primary monoclonal antibody, followed by incubation with HRP-labeled rabbit antimouse secondary antibody. After incubation with AEC chromogen substrate, positive cells with immunostaining spots were visualized under a microscope. (Bottom) Plaque morphology of recombinant hMPVs. An agarose overlay plaque assay was performed in monolayer Vero E6 cells. Viral plaques were developed at day 7 postinfection.

    Techniques Used: Recombinant, Immunostaining, Infection, Incubation, Labeling, Microscopy, Plaque Assay

    21) Product Images from "Fluoxetine Can Inhibit SARS-CoV-2 In Vitro"

    Article Title: Fluoxetine Can Inhibit SARS-CoV-2 In Vitro

    Journal: Microorganisms

    doi: 10.3390/microorganisms9020339

    Levels of infectious particles in cell cultures treated with fluoxetine (FLX) before, during, or after inoculation of SARS-CoV-2. Vero E6 cells were seeded in 24-well plates at 10 5 cells per well. The cells were inoculated with SARS-CoV-2 (black circles), SARS-CoV-2 + 10 μM of FLX (black squares), or SARS-CoV-2 incubated for 2 h and then treated with 10 μM of FLX (white circles), or the cells were incubated for 2 h in presence of 10 μM of FLX, followed by inoculation with SARS-CoV-2 and incubation for 2 h (black triangles). For each condition, the MOI was 0.01 and the cells were washed three times with complete medium after incubation and then treated again with 10 μM of FLX, followed by re-incubation. Levels of infectious particles in supernatants were determined using the endpoint dilution assay at days 1, 2, 3, 5 post-infection. The Spearman–Karber method was used to determine the tissue culture 50% infectious dose (TCID 50 /mL). The results are presented as the mean ± SD of three independent experiments.
    Figure Legend Snippet: Levels of infectious particles in cell cultures treated with fluoxetine (FLX) before, during, or after inoculation of SARS-CoV-2. Vero E6 cells were seeded in 24-well plates at 10 5 cells per well. The cells were inoculated with SARS-CoV-2 (black circles), SARS-CoV-2 + 10 μM of FLX (black squares), or SARS-CoV-2 incubated for 2 h and then treated with 10 μM of FLX (white circles), or the cells were incubated for 2 h in presence of 10 μM of FLX, followed by inoculation with SARS-CoV-2 and incubation for 2 h (black triangles). For each condition, the MOI was 0.01 and the cells were washed three times with complete medium after incubation and then treated again with 10 μM of FLX, followed by re-incubation. Levels of infectious particles in supernatants were determined using the endpoint dilution assay at days 1, 2, 3, 5 post-infection. The Spearman–Karber method was used to determine the tissue culture 50% infectious dose (TCID 50 /mL). The results are presented as the mean ± SD of three independent experiments.

    Techniques Used: Incubation, Endpoint Dilution Assay, Infection

    Levels of infectious particles in supernatants of SARS-CoV-2-infected Vero E6 cells treated daily with Fluoxetine (FLX). Cytopathic effects due to SARS-CoV-2 (MOI 0.01) in Vero E6 cells: ( a ) uninfected Vero E6 cells, ( b ) SARS-CoV-2-infected Vero E6 cells, ( c ) SARS-CoV-2-infected Vero E6 cells treated with 10 μM of FLX. The cells were observed under an inverted microscope (magnification ×100) after 72 h of incubation. ( d ) Vero E6 cells were inoculated with SARS-CoV-2 (MOI 0.01) and treated with 10 μM of FLX. After incubation for 2 h, the cells were washed three times with complete medium and then 10 μM of FLX was added. Cells were treated daily from day 1 to day 7 post-inoculation with 10 μM of FLX. Cell viability was evaluated using the Orangu assay (inverted gray triangles, white circles, dark gray diamonds) and levels of infectious particles in the supernatants were determined using the endpoint dilution assay (gray triangles and black squares) from day 1 through day 9 post-infection. The Spearman–Karber method was used to determine the tissue culture 50% infectious dose (TCID 50 /mL). The results are presented as the mean ± SD of three independent experiments.
    Figure Legend Snippet: Levels of infectious particles in supernatants of SARS-CoV-2-infected Vero E6 cells treated daily with Fluoxetine (FLX). Cytopathic effects due to SARS-CoV-2 (MOI 0.01) in Vero E6 cells: ( a ) uninfected Vero E6 cells, ( b ) SARS-CoV-2-infected Vero E6 cells, ( c ) SARS-CoV-2-infected Vero E6 cells treated with 10 μM of FLX. The cells were observed under an inverted microscope (magnification ×100) after 72 h of incubation. ( d ) Vero E6 cells were inoculated with SARS-CoV-2 (MOI 0.01) and treated with 10 μM of FLX. After incubation for 2 h, the cells were washed three times with complete medium and then 10 μM of FLX was added. Cells were treated daily from day 1 to day 7 post-inoculation with 10 μM of FLX. Cell viability was evaluated using the Orangu assay (inverted gray triangles, white circles, dark gray diamonds) and levels of infectious particles in the supernatants were determined using the endpoint dilution assay (gray triangles and black squares) from day 1 through day 9 post-infection. The Spearman–Karber method was used to determine the tissue culture 50% infectious dose (TCID 50 /mL). The results are presented as the mean ± SD of three independent experiments.

    Techniques Used: Infection, Inverted Microscopy, Incubation, Endpoint Dilution Assay

    Fluoxetine (FLX) can inhibit the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and CV-B4 E2 in Vero E6 cells. Vero E6 cells were seeded in 24-well plates at 10 5 cells per well. ( a ) DMSO or various concentrations of FLX dissolved in DMSO were added to cell cultures to determine the cytotoxic concentrations. Cell viability was assessed using the Orangu assay after 72 h. Optical density values are normalized using the viability value of uninfected cells (mock = 100%). ( b ) Vero E6 cells were inoculated with CV-B4 E2 at MOI 0.01 and various concentrations of FLX dissolved in DMSO were added to cell cultures at non-cytotoxic concentrations; the cells were washed 2 h post-inoculation (DMSO conditions not shown). ( c ) Cell cultures were inoculated with SARS-CoV-2 at MOI 0.01 and 10 μM of FLX; cells were washed 2 h post-inoculation. ( d ) Vero E6 cells were inoculated with SARS-CoV-2 at MOI 0.01 and various concentrations of FLX dissolved in DMSO at non-cytotoxic concentrations. The cells were washed 2 h post-inoculation, and either FLX was added at the same concentration or DMSO (DMSO conditions not shown). Day 2 post-infection, cell viability was expressed as % compared with uninfected FLX-treated cells and levels of infectious particles were determined using the endpoint dilution assay. The Spearman–Karber method was used to determine the tissue culture 50% infectious dose (TCID 50 /mL). The results are expressed as the mean ± SD of three independent experiments.
    Figure Legend Snippet: Fluoxetine (FLX) can inhibit the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and CV-B4 E2 in Vero E6 cells. Vero E6 cells were seeded in 24-well plates at 10 5 cells per well. ( a ) DMSO or various concentrations of FLX dissolved in DMSO were added to cell cultures to determine the cytotoxic concentrations. Cell viability was assessed using the Orangu assay after 72 h. Optical density values are normalized using the viability value of uninfected cells (mock = 100%). ( b ) Vero E6 cells were inoculated with CV-B4 E2 at MOI 0.01 and various concentrations of FLX dissolved in DMSO were added to cell cultures at non-cytotoxic concentrations; the cells were washed 2 h post-inoculation (DMSO conditions not shown). ( c ) Cell cultures were inoculated with SARS-CoV-2 at MOI 0.01 and 10 μM of FLX; cells were washed 2 h post-inoculation. ( d ) Vero E6 cells were inoculated with SARS-CoV-2 at MOI 0.01 and various concentrations of FLX dissolved in DMSO at non-cytotoxic concentrations. The cells were washed 2 h post-inoculation, and either FLX was added at the same concentration or DMSO (DMSO conditions not shown). Day 2 post-infection, cell viability was expressed as % compared with uninfected FLX-treated cells and levels of infectious particles were determined using the endpoint dilution assay. The Spearman–Karber method was used to determine the tissue culture 50% infectious dose (TCID 50 /mL). The results are expressed as the mean ± SD of three independent experiments.

    Techniques Used: Concentration Assay, Infection, Endpoint Dilution Assay

    22) Product Images from "Synthesis of 1-?-D-ribofuranosyl-3-ethynyl-[1,2,4]triazole and its in vitro and in vivo efficacy against Hantavirus"

    Article Title: Synthesis of 1-?-D-ribofuranosyl-3-ethynyl-[1,2,4]triazole and its in vitro and in vivo efficacy against Hantavirus

    Journal:

    doi: 10.1016/j.antiviral.2008.02.003

    Metabolism of ETAR in Vero E6 cells
    Figure Legend Snippet: Metabolism of ETAR in Vero E6 cells

    Techniques Used:

    Dose response of ETAR in Vero E6 cells infected with HTNV or ANDV
    Figure Legend Snippet: Dose response of ETAR in Vero E6 cells infected with HTNV or ANDV

    Techniques Used: Infection

    Effect of ETAR on intracellular GTP levels in Vero E6 cells
    Figure Legend Snippet: Effect of ETAR on intracellular GTP levels in Vero E6 cells

    Techniques Used:

    23) Product Images from "Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis"

    Article Title: Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

    Journal: Journal of Virology

    doi: 10.1128/JVI.03491-13

    siRNAs targeting α5 and αv back cell-cell fusion triggered by hMPV F protein. (A) Knockdown of integrin α5 and αv expression by siRNA. Twenty picomoles of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA was transfected into Vero E6 cells in 24-well plates using Oligofectamine reagents according to the manufacturer's instructions. After 48 h posttransfection, the expression of α5 or αv was detected by Western blotting. (B) Syncytium formation induced by F protein of hMPV after knockdown of integrins α5 and αv. Vero E6 cells in 24-well plates were transfected with 20 pmol of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA. After treatment with siRNAs for 24 h, Vero E6 cells were transfected with 0.8 μg of pCAGGS-F using Lipofectamine Plus reagents and then subjected to pH pulses (pH 5.0). At 48 h, monolayers were fixed with methanol and stained with Giemsa. (C) Quantitation of syncytium formation after knockdown of integrins α5 and αv. The number of syncytia (≥4 nuclei in each syncytium) was counted under a microscope using six randomly selected fields in each siRNA-treated or untreated well. The mean number of syncytia per field was calculated for each treatment. The percent fusion for each siRNA treatment was normalized by the mean number of syncytia in cells transfected with pCAGGS-F without siRNA treatment. The data shown are averages for three independent experiments.
    Figure Legend Snippet: siRNAs targeting α5 and αv back cell-cell fusion triggered by hMPV F protein. (A) Knockdown of integrin α5 and αv expression by siRNA. Twenty picomoles of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA was transfected into Vero E6 cells in 24-well plates using Oligofectamine reagents according to the manufacturer's instructions. After 48 h posttransfection, the expression of α5 or αv was detected by Western blotting. (B) Syncytium formation induced by F protein of hMPV after knockdown of integrins α5 and αv. Vero E6 cells in 24-well plates were transfected with 20 pmol of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA. After treatment with siRNAs for 24 h, Vero E6 cells were transfected with 0.8 μg of pCAGGS-F using Lipofectamine Plus reagents and then subjected to pH pulses (pH 5.0). At 48 h, monolayers were fixed with methanol and stained with Giemsa. (C) Quantitation of syncytium formation after knockdown of integrins α5 and αv. The number of syncytia (≥4 nuclei in each syncytium) was counted under a microscope using six randomly selected fields in each siRNA-treated or untreated well. The mean number of syncytia per field was calculated for each treatment. The percent fusion for each siRNA treatment was normalized by the mean number of syncytia in cells transfected with pCAGGS-F without siRNA treatment. The data shown are averages for three independent experiments.

    Techniques Used: Expressing, Transfection, Western Blot, Staining, Quantitation Assay, Microscopy

    Effects of mutations to the RGD motif on cell-cell fusion triggered by hMPV F protein. (A) Syncytium formation of hMPV F proteins carrying mutations at the RGD motif. Confluent monolayers of Vero E6 cells were transfected with 2 μg plasmids of pCAGGS-F or F-protein mutants. At 24 h posttransfection, the monolayers were fixed with methanol and stained with Giemsa. (B) Content-mixing fusion assay for hMPV F mutants. The extent of fusion for each F mutant was quantitated with the content-mixing fusion assay at pH 5.0 and normalized by the fusion of wild-type hMPV F protein. The data shown are averages for three independent experiments. (C) Cell surface expression of hMPV F mutants. Cell surface expression was determined by FACS using monoclonal antibody against hMPV F protein and normalized by the expression level of wild-type F protein at the cell surface.
    Figure Legend Snippet: Effects of mutations to the RGD motif on cell-cell fusion triggered by hMPV F protein. (A) Syncytium formation of hMPV F proteins carrying mutations at the RGD motif. Confluent monolayers of Vero E6 cells were transfected with 2 μg plasmids of pCAGGS-F or F-protein mutants. At 24 h posttransfection, the monolayers were fixed with methanol and stained with Giemsa. (B) Content-mixing fusion assay for hMPV F mutants. The extent of fusion for each F mutant was quantitated with the content-mixing fusion assay at pH 5.0 and normalized by the fusion of wild-type hMPV F protein. The data shown are averages for three independent experiments. (C) Cell surface expression of hMPV F mutants. Cell surface expression was determined by FACS using monoclonal antibody against hMPV F protein and normalized by the expression level of wild-type F protein at the cell surface.

    Techniques Used: Transfection, Staining, Single Vesicle Fusion Assay, Mutagenesis, Expressing, FACS

    Recovery of recombinant hMPVs carrying mutations in the RGD motif. (Top) Immunostaining spots formed by recombinant hMPVs. LLC-MK2 cells were infected with recombinant hMPV mutants and incubated at 37°C for 1 h. At day 4 postinfection, the supernatant was removed and cells were fixed. The cells were then labeled with an anti-hMPV N protein primary monoclonal antibody, followed by incubation with HRP-labeled rabbit antimouse secondary antibody. After incubation with AEC chromogen substrate, positive cells with immunostaining spots were visualized under a microscope. (Bottom) Plaque morphology of recombinant hMPVs. An agarose overlay plaque assay was performed in monolayer Vero E6 cells. Viral plaques were developed at day 7 postinfection.
    Figure Legend Snippet: Recovery of recombinant hMPVs carrying mutations in the RGD motif. (Top) Immunostaining spots formed by recombinant hMPVs. LLC-MK2 cells were infected with recombinant hMPV mutants and incubated at 37°C for 1 h. At day 4 postinfection, the supernatant was removed and cells were fixed. The cells were then labeled with an anti-hMPV N protein primary monoclonal antibody, followed by incubation with HRP-labeled rabbit antimouse secondary antibody. After incubation with AEC chromogen substrate, positive cells with immunostaining spots were visualized under a microscope. (Bottom) Plaque morphology of recombinant hMPVs. An agarose overlay plaque assay was performed in monolayer Vero E6 cells. Viral plaques were developed at day 7 postinfection.

    Techniques Used: Recombinant, Immunostaining, Infection, Incubation, Labeling, Microscopy, Plaque Assay

    24) Product Images from "Standardization of the Filovirus Plaque Assay for Use in Preclinical Studies"

    Article Title: Standardization of the Filovirus Plaque Assay for Use in Preclinical Studies

    Journal: Viruses

    doi: 10.3390/v4123511

    Filovirus plaques produced on Vero E6 cells from two sources are similar in appearance and titer. ( A ) EBOV plaques on ATCC Vero E6 cells plated at (1) 24 hours before assay, and (2) 72 hours before assay. EBOV plaques on BEI Vero E6 cells plated at (3) 24 hours before assay, and (4) 72 hours before assay. (5) MARV plaques on Vero cells from (5) ATCC and (6) BEI plated 24 hours before assay. ( B ) EBOV titers are similar in Vero E6 cells from ATCC and BEI when measured independently by two operators. This experiment was performed twice with up to 3 operators (data not shown), and one representative graph is shown. Each bar represents an average of 7 replicates.
    Figure Legend Snippet: Filovirus plaques produced on Vero E6 cells from two sources are similar in appearance and titer. ( A ) EBOV plaques on ATCC Vero E6 cells plated at (1) 24 hours before assay, and (2) 72 hours before assay. EBOV plaques on BEI Vero E6 cells plated at (3) 24 hours before assay, and (4) 72 hours before assay. (5) MARV plaques on Vero cells from (5) ATCC and (6) BEI plated 24 hours before assay. ( B ) EBOV titers are similar in Vero E6 cells from ATCC and BEI when measured independently by two operators. This experiment was performed twice with up to 3 operators (data not shown), and one representative graph is shown. Each bar represents an average of 7 replicates.

    Techniques Used: Produced

    Vero E6 cells are suitable for quantitation of EBOV plaques. ( A ) Vero and Vero E6 cells produce EBOV plaques. ( B ) EBOV titers are similar in ATCC Vero E6 cells plated 24, 48 or 72 hours prior to plaque assay. This experiment was performed twice, and one representative graph is shown. Each bar represents an average of 5 replicates. The * indicates p = 0.006 between 24 and 72 hour samples for passage 29.
    Figure Legend Snippet: Vero E6 cells are suitable for quantitation of EBOV plaques. ( A ) Vero and Vero E6 cells produce EBOV plaques. ( B ) EBOV titers are similar in ATCC Vero E6 cells plated 24, 48 or 72 hours prior to plaque assay. This experiment was performed twice, and one representative graph is shown. Each bar represents an average of 5 replicates. The * indicates p = 0.006 between 24 and 72 hour samples for passage 29.

    Techniques Used: Quantitation Assay, Plaque Assay

    ( A ) EBOV titers in ATCC or BEI Vero E6 cells of various passage ages. This experiment was performed by three independent investigators, four replicates per cell type. The combined data are presented, where each bar represents 12 replicates. * indicates a significant difference in value between BEI passage 40 and passage 27 ( p = 0.0003), ** a difference between ATCC passage 54 and 28 ( p = 0.007), and *** a difference between BEI passage 40 and ATCC passage 28 ( p = 0.000002). For these experiments, p value cutoff was ≤0.008). ( B ) Analysis of EBOV titer changes in cells of various passages. The arrows point out data from passages plotted in (A).
    Figure Legend Snippet: ( A ) EBOV titers in ATCC or BEI Vero E6 cells of various passage ages. This experiment was performed by three independent investigators, four replicates per cell type. The combined data are presented, where each bar represents 12 replicates. * indicates a significant difference in value between BEI passage 40 and passage 27 ( p = 0.0003), ** a difference between ATCC passage 54 and 28 ( p = 0.007), and *** a difference between BEI passage 40 and ATCC passage 28 ( p = 0.000002). For these experiments, p value cutoff was ≤0.008). ( B ) Analysis of EBOV titer changes in cells of various passages. The arrows point out data from passages plotted in (A).

    Techniques Used:

    25) Product Images from "Immunization with an Attenuated Severe Acute Respiratory Syndrome Coronavirus Deleted in E Protein Protects Against Lethal Respiratory Disease"

    Article Title: Immunization with an Attenuated Severe Acute Respiratory Syndrome Coronavirus Deleted in E Protein Protects Against Lethal Respiratory Disease

    Journal: Virology

    doi: 10.1016/j.virol.2010.01.004

    Clinical effects and vaccine efficacy of rSARS-CoV-ΔE and rSARS-CoV-Δ[E,6-9b] after 16 passages through Vero E6 cells (A, B). 6 week old hACE2 Tg mice were inoculated with 12,000 PFU of rSARS-CoV-ΔE-p16 (circles, n = 14) or rSARS-CoV-Δ[E,6-9b]-p16 (squares, n = 18) or wild type rSARS-CoV (triangles, n = 4) and monitored daily for survival (A) and weight loss (B). Differences in survival were statistically significant (P
    Figure Legend Snippet: Clinical effects and vaccine efficacy of rSARS-CoV-ΔE and rSARS-CoV-Δ[E,6-9b] after 16 passages through Vero E6 cells (A, B). 6 week old hACE2 Tg mice were inoculated with 12,000 PFU of rSARS-CoV-ΔE-p16 (circles, n = 14) or rSARS-CoV-Δ[E,6-9b]-p16 (squares, n = 18) or wild type rSARS-CoV (triangles, n = 4) and monitored daily for survival (A) and weight loss (B). Differences in survival were statistically significant (P

    Techniques Used: Mouse Assay

    26) Product Images from "Severe Acute Respiratory Syndrome-Related Coronavirus Is Inhibited by Interferon-α"

    Article Title: Severe Acute Respiratory Syndrome-Related Coronavirus Is Inhibited by Interferon-α

    Journal: The Journal of Infectious Diseases

    doi: 10.1086/382597

    Effect of interferon (IFN)-α-2b on virus growth. A , Reduction of virus yield in the presence of antiviral activity. Vero E6 cells were infected with ∼100 pfu of the severe acute respiratory syndrome-related coronavirus (SARS CoV). At 1 h after infection, the supernatants were replaced with Dulbecco's modified Eagle's medium (DMEM) or DMEM containing the indicated amounts of IFN-α-2b (IU/mL) for a total of 72 h. Growth of the SARS CoV isolate Tor2 was determined by plaquetitration assay from 3 independent experiments, and data are mean ± SD. B , Analysis of cell-to-cell spread of SARS CoV in the presence and absence of IFN-α-2b. Vero E6 cell monolayers were infected with 10-fold dilutions of the SARS CoV. After 1 h of virus adsorption, the inoculum was removed, and cell monolayers were overlaid with 0.9% low-meltingpoint agarose in DMEM (B2) or DMEM containing 5000 IU/mL of IFNa- 2b (B1). On day 3 after infection, cells were stained with crystal violet. C , Protein analysis. Cell lysates were subjected to 10% SDS-PAGE. Protein was electrotransferred to polyvinylidene difluoride membrane (Immobilon P membrane; Millipore). Viral antigen was detected by use of a patient serum sample and horseradish peroxidase-conjugated anti-human IgG, by use of enhanced chemiluminescence. NP, nucleoprotein. D , Comparison of the effect of antiviral activity on different SARS CoV isolates. Vero E6 cells were infected with 4 independently isolated SARS CoVs (isolates Tor2, Tor3, Tor7, and Tor684) and were analyzed for their sensitivity to IFN-α-2b, as described above.
    Figure Legend Snippet: Effect of interferon (IFN)-α-2b on virus growth. A , Reduction of virus yield in the presence of antiviral activity. Vero E6 cells were infected with ∼100 pfu of the severe acute respiratory syndrome-related coronavirus (SARS CoV). At 1 h after infection, the supernatants were replaced with Dulbecco's modified Eagle's medium (DMEM) or DMEM containing the indicated amounts of IFN-α-2b (IU/mL) for a total of 72 h. Growth of the SARS CoV isolate Tor2 was determined by plaquetitration assay from 3 independent experiments, and data are mean ± SD. B , Analysis of cell-to-cell spread of SARS CoV in the presence and absence of IFN-α-2b. Vero E6 cell monolayers were infected with 10-fold dilutions of the SARS CoV. After 1 h of virus adsorption, the inoculum was removed, and cell monolayers were overlaid with 0.9% low-meltingpoint agarose in DMEM (B2) or DMEM containing 5000 IU/mL of IFNa- 2b (B1). On day 3 after infection, cells were stained with crystal violet. C , Protein analysis. Cell lysates were subjected to 10% SDS-PAGE. Protein was electrotransferred to polyvinylidene difluoride membrane (Immobilon P membrane; Millipore). Viral antigen was detected by use of a patient serum sample and horseradish peroxidase-conjugated anti-human IgG, by use of enhanced chemiluminescence. NP, nucleoprotein. D , Comparison of the effect of antiviral activity on different SARS CoV isolates. Vero E6 cells were infected with 4 independently isolated SARS CoVs (isolates Tor2, Tor3, Tor7, and Tor684) and were analyzed for their sensitivity to IFN-α-2b, as described above.

    Techniques Used: Activity Assay, Infection, Modification, Adsorption, Staining, SDS Page, Isolation

    Cytopathic effect in severe acute respiratory syndrome-related coronavirus (SARS CoV)-infected Vero E6 cells. Vero E6 cells were infected with the SARS CoV at an MOI of 0.001 for 1 h and then were incubated in Dulbecco's modified Eagle's medium containing the indicated amounts of interferon-α-2b (IU/mL). Cells were fixed with 4% formalin on day 3 after infection.
    Figure Legend Snippet: Cytopathic effect in severe acute respiratory syndrome-related coronavirus (SARS CoV)-infected Vero E6 cells. Vero E6 cells were infected with the SARS CoV at an MOI of 0.001 for 1 h and then were incubated in Dulbecco's modified Eagle's medium containing the indicated amounts of interferon-α-2b (IU/mL). Cells were fixed with 4% formalin on day 3 after infection.

    Techniques Used: Infection, Incubation, Modification

    27) Product Images from "In-Cell Western Assays to Evaluate Hantaan Virus Replication as a Novel Approach to Screen Antiviral Molecules and Detect Neutralizing Antibody Titers"

    Article Title: In-Cell Western Assays to Evaluate Hantaan Virus Replication as a Novel Approach to Screen Antiviral Molecules and Detect Neutralizing Antibody Titers

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2017.00269

    Application of the ICW assay to detect the HTNV titers HUVECs were seeded into ten 96-well microplates of different brands (Falcon™ for microplates numbered 1–5 and Nunc™ numbered 6–10) and were mock infected (negative control, NC) or infected with HTNV at an MOI of 0.1 (positive control, PC). The ICW assay was performed to observe and calculate the amount of HTNV NP production ( A and C for the NC group and B and D for the PC group). The P/N value was determined by setting the intensity ratio from the number 1 microplate as the calibrator (E) . HTNV propagated in Vero E6 cells was serially diluted from 1:10 to 1:10 6 and used to infect A549 cells (F) or E6 cells (H) in 96-well microplates. A549 cells were acquired for the ICW assay at 2 dpi (F) , and E6 cells were collected for ELISA at 10 dpi (H) . The P/N value was calculated by ICW (G) and ELISA (I) , and the viral titer was determined by TCID50 with the Reed and Muench formula. HTNV was propagated in mouse brains in five independent experiments and propagated in Vero E6 cells. The ten batches of HTNV were used for titer assessment by both ICW and ELISA. The relationship between the ICW-derived and ELISA-derived titers was analyzed using the rank correlation test (J) . Data are presented as the mean ± SD.
    Figure Legend Snippet: Application of the ICW assay to detect the HTNV titers HUVECs were seeded into ten 96-well microplates of different brands (Falcon™ for microplates numbered 1–5 and Nunc™ numbered 6–10) and were mock infected (negative control, NC) or infected with HTNV at an MOI of 0.1 (positive control, PC). The ICW assay was performed to observe and calculate the amount of HTNV NP production ( A and C for the NC group and B and D for the PC group). The P/N value was determined by setting the intensity ratio from the number 1 microplate as the calibrator (E) . HTNV propagated in Vero E6 cells was serially diluted from 1:10 to 1:10 6 and used to infect A549 cells (F) or E6 cells (H) in 96-well microplates. A549 cells were acquired for the ICW assay at 2 dpi (F) , and E6 cells were collected for ELISA at 10 dpi (H) . The P/N value was calculated by ICW (G) and ELISA (I) , and the viral titer was determined by TCID50 with the Reed and Muench formula. HTNV was propagated in mouse brains in five independent experiments and propagated in Vero E6 cells. The ten batches of HTNV were used for titer assessment by both ICW and ELISA. The relationship between the ICW-derived and ELISA-derived titers was analyzed using the rank correlation test (J) . Data are presented as the mean ± SD.

    Techniques Used: Infection, Negative Control, Positive Control, Enzyme-linked Immunosorbent Assay, Derivative Assay

    28) Product Images from "Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis"

    Article Title: Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis

    Journal: Journal of Virology

    doi: 10.1128/JVI.00755-16

    Multistep growth curve of recombinant hMPVs carrying mutations in the phosphorylation site. Vero E6 cells in 35-mm dishes were infected with each recombinant hMPV at an MOI of 0.01. After adsorption for 1 h, the inocula were removed and the infected cells were washed 3 times with Opti-MEM. Fresh Opti-MEM containing 2% FBS then was added and cells were incubated at 37°C for various time periods. Aliquots of the cell culture fluid were removed at the indicated intervals. Viral titer was determined by an immunostaining assay in Vero E6 cells. Viral titers of each mutant at each time point were compared to those of rhMPV (*, P
    Figure Legend Snippet: Multistep growth curve of recombinant hMPVs carrying mutations in the phosphorylation site. Vero E6 cells in 35-mm dishes were infected with each recombinant hMPV at an MOI of 0.01. After adsorption for 1 h, the inocula were removed and the infected cells were washed 3 times with Opti-MEM. Fresh Opti-MEM containing 2% FBS then was added and cells were incubated at 37°C for various time periods. Aliquots of the cell culture fluid were removed at the indicated intervals. Viral titer was determined by an immunostaining assay in Vero E6 cells. Viral titers of each mutant at each time point were compared to those of rhMPV (*, P

    Techniques Used: Recombinant, Infection, Adsorption, Incubation, Cell Culture, Immunostaining, Mutagenesis

    Recovery of recombinant hMPVs carrying mutations in the phosphorylation sites of M2-1. (A) Immunostaining spots formed by recombinant hMPVs. Vero E6 cells were infected with recombinant hMPV mutants and incubated at 37°C for 5 days. The cells were stained with an anti-hMPV N protein monoclonal antibody. (B) Plaque morphology of recombinant hMPVs. An agarose overlay plaque assay was performed on a monolayer of Vero E6 cells. Viral plaques were developed at day 8 postinfection. The plaque pictures were taken from different dilutions of each hMPV mutant. Data were averages from 20 plaques ± 1 standard deviation.
    Figure Legend Snippet: Recovery of recombinant hMPVs carrying mutations in the phosphorylation sites of M2-1. (A) Immunostaining spots formed by recombinant hMPVs. Vero E6 cells were infected with recombinant hMPV mutants and incubated at 37°C for 5 days. The cells were stained with an anti-hMPV N protein monoclonal antibody. (B) Plaque morphology of recombinant hMPVs. An agarose overlay plaque assay was performed on a monolayer of Vero E6 cells. Viral plaques were developed at day 8 postinfection. The plaque pictures were taken from different dilutions of each hMPV mutant. Data were averages from 20 plaques ± 1 standard deviation.

    Techniques Used: Recombinant, Immunostaining, Infection, Incubation, Staining, Plaque Assay, Mutagenesis, Standard Deviation

    29) Product Images from "Ribavirin Causes Error Catastrophe during Hantaan Virus Replication"

    Article Title: Ribavirin Causes Error Catastrophe during Hantaan Virus Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.1.481-488.2003

    Effect of ribavirin on the yield of infectious HTNV. Supernatants were harvested from HTNV-infected cells at MOIs of 0.01 (A) and 0.1 (B) for the various treatments, i.e., 0 (•) or 24 (▪) μg of ribavirin, and then assayed for plaque formation on monolayers of Vero E6 cells.
    Figure Legend Snippet: Effect of ribavirin on the yield of infectious HTNV. Supernatants were harvested from HTNV-infected cells at MOIs of 0.01 (A) and 0.1 (B) for the various treatments, i.e., 0 (•) or 24 (▪) μg of ribavirin, and then assayed for plaque formation on monolayers of Vero E6 cells.

    Techniques Used: Infection

    30) Product Images from "Identification of Novel Antiviral Compounds Targeting Entry of Hantaviruses"

    Article Title: Identification of Novel Antiviral Compounds Targeting Entry of Hantaviruses

    Journal: Viruses

    doi: 10.3390/v13040685

    Performance of EGFP expression measurement as a method to differentiate non-treated from treated infected cells. ( A ) Representative image of a 96-well plate with two enlarged wells standing for treatment conditions. Three conditions were applied to confluent monolayers of A549 cells: no pre-treatment and no infection; no pre-treatment followed by infection with VSV*ΔG-Luc(HTNV-G) (upper right panel); or pre-treatment with 50 μM EIPA for 30 min (lower right panel), followed by infection with VSV*ΔG-Luc(HTNV-G) in the presence of the inhibitor. After 16 h of incubation, cells were washed, fixed with 2% PFA, and the nuclei were stained (DAPI). The number of infected cells was then analyzed through GFP expression using fluorescent microscope. Representation of EGFP expression on the XLS microscope. ( B , C ) ROC curves obtained for non-infected vs. infected cells ( B ) and for non-treated vs. EIPA-treated cells ( C ). The AUC and significance level are shown in each case. ( D ) EIPA inhibits HTNV-G in Vero E6 and A549 cells. Monolayers of Vero E6 or A549 cells were pre-treated with indicated concentration of EIPA for 45 min at 37 °C, followed by VSV*ΔG-Luc(HTNV-G) infection at 200 IU/well for 90 min. Cells were then washed and complete medium with 20 mM ammonium chloride was added and incubated for 16 h. The infection levels were assessed by counting the EGFP-positive infected cells. Data are means + SD ( n = 3). ( E ) EIPA inhibits HTNV infection. Vero E6 cells were pre-incubated with EIPA at the indicated concentrations for 45 min at 37 °C, followed by HTNV infection for 1 h at 37 °C. Cells were subsequently washed once with medium and incubated with medium containing appropriate drug concentration for 48 h. Infection levels were assessed by RT-qPCR. Data are means + SD ( n = 3) of genome per mL.
    Figure Legend Snippet: Performance of EGFP expression measurement as a method to differentiate non-treated from treated infected cells. ( A ) Representative image of a 96-well plate with two enlarged wells standing for treatment conditions. Three conditions were applied to confluent monolayers of A549 cells: no pre-treatment and no infection; no pre-treatment followed by infection with VSV*ΔG-Luc(HTNV-G) (upper right panel); or pre-treatment with 50 μM EIPA for 30 min (lower right panel), followed by infection with VSV*ΔG-Luc(HTNV-G) in the presence of the inhibitor. After 16 h of incubation, cells were washed, fixed with 2% PFA, and the nuclei were stained (DAPI). The number of infected cells was then analyzed through GFP expression using fluorescent microscope. Representation of EGFP expression on the XLS microscope. ( B , C ) ROC curves obtained for non-infected vs. infected cells ( B ) and for non-treated vs. EIPA-treated cells ( C ). The AUC and significance level are shown in each case. ( D ) EIPA inhibits HTNV-G in Vero E6 and A549 cells. Monolayers of Vero E6 or A549 cells were pre-treated with indicated concentration of EIPA for 45 min at 37 °C, followed by VSV*ΔG-Luc(HTNV-G) infection at 200 IU/well for 90 min. Cells were then washed and complete medium with 20 mM ammonium chloride was added and incubated for 16 h. The infection levels were assessed by counting the EGFP-positive infected cells. Data are means + SD ( n = 3). ( E ) EIPA inhibits HTNV infection. Vero E6 cells were pre-incubated with EIPA at the indicated concentrations for 45 min at 37 °C, followed by HTNV infection for 1 h at 37 °C. Cells were subsequently washed once with medium and incubated with medium containing appropriate drug concentration for 48 h. Infection levels were assessed by RT-qPCR. Data are means + SD ( n = 3) of genome per mL.

    Techniques Used: Expressing, Infection, Incubation, Staining, Microscopy, Concentration Assay, Quantitative RT-PCR

    Treatment with tetrandrine, monensin sodium salt and emetine dihydrochloride in Vero E6 cells. ( A – C ) Monolayers of Vero E6 cells were pre-treated with the indicated drugs at given concentrations and then infected with VSV*ΔG-Luc(HTNV-G). After 90 min, cells were washed and complete medium with 20 mM ammonium chloride was added and further incubated for 16 h. The infection levels were assessed by counting the EGFP-positive infected cells. Data are means + SD ( n = 3).
    Figure Legend Snippet: Treatment with tetrandrine, monensin sodium salt and emetine dihydrochloride in Vero E6 cells. ( A – C ) Monolayers of Vero E6 cells were pre-treated with the indicated drugs at given concentrations and then infected with VSV*ΔG-Luc(HTNV-G). After 90 min, cells were washed and complete medium with 20 mM ammonium chloride was added and further incubated for 16 h. The infection levels were assessed by counting the EGFP-positive infected cells. Data are means + SD ( n = 3).

    Techniques Used: Infection, Incubation

    Validation with authentic pathogenic viruses. ( A , B ) Vero E6 cells were pre-incubated with emetine dihydrochloride (panel A), or tetrandrine (panel B) at the indicated concentrations for 45 min at 37 °C, followed by HTNV infection for 1 h at 37 °C. Cells were subsequently washed once with medium and incubated with medium containing appropriate drug concentrations for 48 h. Infection levels were assessed by RT-qPCR. Data are means + SD ( n = 3) of genome per ml. ( C ) Cell viability over two days. Monolayers of Vero E6 cells were treated with the indicated concentration of emetine dihydrochloride or tetrandrine under the assay conditions ( A , B ). After 48 h, intracellular ATP levels were measured using CellTiter-Glo ® assay. Data are means ± SD ( n = 3) of relative light units (RLU). ( D ) Calculation of the therapeutic index (TI). The therapeutic index is defined as toxic dose (TD 50 )/effective dose (ED 50 ).
    Figure Legend Snippet: Validation with authentic pathogenic viruses. ( A , B ) Vero E6 cells were pre-incubated with emetine dihydrochloride (panel A), or tetrandrine (panel B) at the indicated concentrations for 45 min at 37 °C, followed by HTNV infection for 1 h at 37 °C. Cells were subsequently washed once with medium and incubated with medium containing appropriate drug concentrations for 48 h. Infection levels were assessed by RT-qPCR. Data are means + SD ( n = 3) of genome per ml. ( C ) Cell viability over two days. Monolayers of Vero E6 cells were treated with the indicated concentration of emetine dihydrochloride or tetrandrine under the assay conditions ( A , B ). After 48 h, intracellular ATP levels were measured using CellTiter-Glo ® assay. Data are means ± SD ( n = 3) of relative light units (RLU). ( D ) Calculation of the therapeutic index (TI). The therapeutic index is defined as toxic dose (TD 50 )/effective dose (ED 50 ).

    Techniques Used: Incubation, Infection, Quantitative RT-PCR, Concentration Assay, Glo Assay

    31) Product Images from "Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes in COVID-19 convalescent plasma"

    Article Title: Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes in COVID-19 convalescent plasma

    Journal: bioRxiv

    doi: 10.1101/2020.12.20.423708

    Independent live virus neutralization titers of recombinant plasma IgG mAbs CM17, CM25, and CM32. In vitro live virus neutralization curves for CM17, CM25, and CM32 repeated in second independent laboratory demonstrate similar levels of inhibition (as compared to data in Fig.1e and 2c) of live SARS-CoV-2 virus infection of monolayered Vero E6 cells. The percent of infected Vero E6 cells in each sample dilution was normalized relative to the virus-only (no plasma) negative control sample.
    Figure Legend Snippet: Independent live virus neutralization titers of recombinant plasma IgG mAbs CM17, CM25, and CM32. In vitro live virus neutralization curves for CM17, CM25, and CM32 repeated in second independent laboratory demonstrate similar levels of inhibition (as compared to data in Fig.1e and 2c) of live SARS-CoV-2 virus infection of monolayered Vero E6 cells. The percent of infected Vero E6 cells in each sample dilution was normalized relative to the virus-only (no plasma) negative control sample.

    Techniques Used: Neutralization, Recombinant, In Vitro, Inhibition, Infection, Negative Control

    Live virus neutralization titers of four COVID+ study subjects’ plasma at each collection time point. Serial dilutions of plasma were tested in duplicate (SD error bars) for inhibition of live SARS-CoV-2 virus infection of in vitro monolayered Vero E6 cells. The percent of infected Vero E6 cells in each sample dilution was normalized relative to the virus-only (no plasma) negative control sample.
    Figure Legend Snippet: Live virus neutralization titers of four COVID+ study subjects’ plasma at each collection time point. Serial dilutions of plasma were tested in duplicate (SD error bars) for inhibition of live SARS-CoV-2 virus infection of in vitro monolayered Vero E6 cells. The percent of infected Vero E6 cells in each sample dilution was normalized relative to the virus-only (no plasma) negative control sample.

    Techniques Used: Neutralization, Inhibition, Infection, In Vitro, Negative Control

    32) Product Images from "Methyltransferase-Defective Avian Metapneumovirus Vaccines Provide Complete Protection against Challenge with the Homologous Colorado Strain and the Heterologous Minnesota Strain"

    Article Title: Methyltransferase-Defective Avian Metapneumovirus Vaccines Provide Complete Protection against Challenge with the Homologous Colorado Strain and the Heterologous Minnesota Strain

    Journal: Journal of Virology

    doi: 10.1128/JVI.01095-14

    Plaque morphology of recombinant aMPVs carrying mutations in the SAM binding site. An agarose overlay plaque assay was performed in monolayer Vero-E6 cells. Viral plaques were developed at day 7 postinfection. The cells were fixed in 10% formaldehyde,
    Figure Legend Snippet: Plaque morphology of recombinant aMPVs carrying mutations in the SAM binding site. An agarose overlay plaque assay was performed in monolayer Vero-E6 cells. Viral plaques were developed at day 7 postinfection. The cells were fixed in 10% formaldehyde,

    Techniques Used: Recombinant, Binding Assay, Plaque Assay

    Multistep growth curve of recombinant hMPVs carrying mutations in the SAM binding site. Vero-E6 cells in 35-mm dishes were infected with each recombinant raMPV at an MOI of 0.1. After adsorption for 1 h, the inocula were removed, and the infected cells
    Figure Legend Snippet: Multistep growth curve of recombinant hMPVs carrying mutations in the SAM binding site. Vero-E6 cells in 35-mm dishes were infected with each recombinant raMPV at an MOI of 0.1. After adsorption for 1 h, the inocula were removed, and the infected cells

    Techniques Used: Recombinant, Binding Assay, Infection, Adsorption

    Cytopathic effects (CPEs) caused by raMPVs carrying mutations in the SAM binding site. Vero-E6 cells were infected with each recombinant aMPV at an MOI of 0.1. CPE was monitored on a daily basis. Pictures were taken at days 2, 4, and 6 postinfection.
    Figure Legend Snippet: Cytopathic effects (CPEs) caused by raMPVs carrying mutations in the SAM binding site. Vero-E6 cells were infected with each recombinant aMPV at an MOI of 0.1. CPE was monitored on a daily basis. Pictures were taken at days 2, 4, and 6 postinfection.

    Techniques Used: Binding Assay, Infection, Recombinant

    33) Product Images from "Dynein-Dependent Transport of the Hantaan Virus Nucleocapsid Protein to the Endoplasmic Reticulum-Golgi Intermediate Compartment ▿"

    Article Title: Dynein-Dependent Transport of the Hantaan Virus Nucleocapsid Protein to the Endoplasmic Reticulum-Golgi Intermediate Compartment ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00418-07

    Distribution of N in Vero E6 cells and redistribution upon treatment with CytD, BFA, or NOC. Vero E6 cells were transfected with pcHTNVN, which expresses N, and after 18 h the cells were treated with a mock vector (A), CytD (B), BFA (C), or NOC (D). Slides
    Figure Legend Snippet: Distribution of N in Vero E6 cells and redistribution upon treatment with CytD, BFA, or NOC. Vero E6 cells were transfected with pcHTNVN, which expresses N, and after 18 h the cells were treated with a mock vector (A), CytD (B), BFA (C), or NOC (D). Slides

    Techniques Used: Transfection, Plasmid Preparation

    Redistribution of N in HTNV-infected Vero E6 cells and redistribution upon treatment with CytD, BFA, or NOC. Vero E6 cells were infected with HTNV at an MOI of 0.1, and after 3 days the cells were treated for 1 h with a mock vector (A), CytD (B), BFA
    Figure Legend Snippet: Redistribution of N in HTNV-infected Vero E6 cells and redistribution upon treatment with CytD, BFA, or NOC. Vero E6 cells were infected with HTNV at an MOI of 0.1, and after 3 days the cells were treated for 1 h with a mock vector (A), CytD (B), BFA

    Techniques Used: Infection, Plasmid Preparation

    Colocalization of HTNV N with ERGIC-53 and redistribution of N with BFA. Vero E6 cells were infected with HTNV at an MOI of 0.1, and after 3 days slides were acetone fixed (except for Mann II staining, in which case paraformaldehyde was used). Prior to
    Figure Legend Snippet: Colocalization of HTNV N with ERGIC-53 and redistribution of N with BFA. Vero E6 cells were infected with HTNV at an MOI of 0.1, and after 3 days slides were acetone fixed (except for Mann II staining, in which case paraformaldehyde was used). Prior to

    Techniques Used: Infection, Staining

    Relationship of N and vimentin in HTNV-infected and pcHTNVN-transfected Vero E6 cells. Vero E6 cells were infected with HTNV at an MOI of 0.1 (A) or were transfected with pcHTNVN (B), and after 5 days or 24 h, respectively, slides were acetone fixed and
    Figure Legend Snippet: Relationship of N and vimentin in HTNV-infected and pcHTNVN-transfected Vero E6 cells. Vero E6 cells were infected with HTNV at an MOI of 0.1 (A) or were transfected with pcHTNVN (B), and after 5 days or 24 h, respectively, slides were acetone fixed and

    Techniques Used: Infection, Transfection

    Temporal distribution of N in Vero E6 cells infected with HTNV or transfected with plasmid expressing N. (A) To ensure maximal infection, Vero E6 cells were infected with HTNV at an MOI of 5.0 and were examined for the presence of N at 4, 12, 24, and
    Figure Legend Snippet: Temporal distribution of N in Vero E6 cells infected with HTNV or transfected with plasmid expressing N. (A) To ensure maximal infection, Vero E6 cells were infected with HTNV at an MOI of 5.0 and were examined for the presence of N at 4, 12, 24, and

    Techniques Used: Infection, Transfection, Plasmid Preparation, Expressing

    Colocalization studies of HTNV N in Vero E6 cells expressing N alone, as well as redistribution of N with BFA, with various subcellular markers. Vero E6 cells were transfected with pcHTNVN, which expresses N alone, and after 18 h, the cells were fixed
    Figure Legend Snippet: Colocalization studies of HTNV N in Vero E6 cells expressing N alone, as well as redistribution of N with BFA, with various subcellular markers. Vero E6 cells were transfected with pcHTNVN, which expresses N alone, and after 18 h, the cells were fixed

    Techniques Used: Expressing, Transfection

    Association of HTNV N with membrane fractions. Vero E6 cells were transfected with pcHTNVN, and after 18 h they were subjected to membrane floatation (A) or subcellular fractionation (B). Fractions were subjected to Western blotting and were probed with
    Figure Legend Snippet: Association of HTNV N with membrane fractions. Vero E6 cells were transfected with pcHTNVN, and after 18 h they were subjected to membrane floatation (A) or subcellular fractionation (B). Fractions were subjected to Western blotting and were probed with

    Techniques Used: Transfection, Fractionation, Western Blot

    Overexpression of dynamitin abrogated accumulation of N in the perinuclear region. Vero E6 cells were cotransfected with p50-GFP (green) and pcHTNVN (red). (A) Vero E6 cells infected with HTNV are shown, either mock treated (left panel), treated for 1
    Figure Legend Snippet: Overexpression of dynamitin abrogated accumulation of N in the perinuclear region. Vero E6 cells were cotransfected with p50-GFP (green) and pcHTNVN (red). (A) Vero E6 cells infected with HTNV are shown, either mock treated (left panel), treated for 1

    Techniques Used: Over Expression, Infection

    34) Product Images from "Immunization with an Attenuated Severe Acute Respiratory Syndrome Coronavirus Deleted in E Protein Protects Against Lethal Respiratory Disease"

    Article Title: Immunization with an Attenuated Severe Acute Respiratory Syndrome Coronavirus Deleted in E Protein Protects Against Lethal Respiratory Disease

    Journal: Virology

    doi: 10.1016/j.virol.2010.01.004

    Clinical effects and vaccine efficacy of rSARS-CoV-ΔE and rSARS-CoV-Δ[E,6-9b] after 16 passages through Vero E6 cells (A, B). 6 week old hACE2 Tg mice were inoculated with 12,000 PFU of rSARS-CoV-ΔE-p16 (circles, n = 14) or rSARS-CoV-Δ[E,6-9b]-p16 (squares, n = 18) or wild type rSARS-CoV (triangles, n = 4) and monitored daily for survival (A) and weight loss (B). Differences in survival were statistically significant (P
    Figure Legend Snippet: Clinical effects and vaccine efficacy of rSARS-CoV-ΔE and rSARS-CoV-Δ[E,6-9b] after 16 passages through Vero E6 cells (A, B). 6 week old hACE2 Tg mice were inoculated with 12,000 PFU of rSARS-CoV-ΔE-p16 (circles, n = 14) or rSARS-CoV-Δ[E,6-9b]-p16 (squares, n = 18) or wild type rSARS-CoV (triangles, n = 4) and monitored daily for survival (A) and weight loss (B). Differences in survival were statistically significant (P

    Techniques Used: Mouse Assay

    35) Product Images from "Severe Acute Respiratory Syndrome-Related Coronavirus Is Inhibited by Interferon-α"

    Article Title: Severe Acute Respiratory Syndrome-Related Coronavirus Is Inhibited by Interferon-α

    Journal: The Journal of Infectious Diseases

    doi: 10.1086/382597

    Effect of interferon (IFN)-α-2b on virus growth. A , Reduction of virus yield in the presence of antiviral activity. Vero E6 cells were infected with ∼100 pfu of the severe acute respiratory syndrome-related coronavirus (SARS CoV). At 1 h after infection, the supernatants were replaced with Dulbecco's modified Eagle's medium (DMEM) or DMEM containing the indicated amounts of IFN-α-2b (IU/mL) for a total of 72 h. Growth of the SARS CoV isolate Tor2 was determined by plaquetitration assay from 3 independent experiments, and data are mean ± SD. B , Analysis of cell-to-cell spread of SARS CoV in the presence and absence of IFN-α-2b. Vero E6 cell monolayers were infected with 10-fold dilutions of the SARS CoV. After 1 h of virus adsorption, the inoculum was removed, and cell monolayers were overlaid with 0.9% low-meltingpoint agarose in DMEM (B2) or DMEM containing 5000 IU/mL of IFNa- 2b (B1). On day 3 after infection, cells were stained with crystal violet. C , Protein analysis. Cell lysates were subjected to 10% SDS-PAGE. Protein was electrotransferred to polyvinylidene difluoride membrane (Immobilon P membrane; Millipore). Viral antigen was detected by use of a patient serum sample and horseradish peroxidase-conjugated anti-human IgG, by use of enhanced chemiluminescence. NP, nucleoprotein. D , Comparison of the effect of antiviral activity on different SARS CoV isolates. Vero E6 cells were infected with 4 independently isolated SARS CoVs (isolates Tor2, Tor3, Tor7, and Tor684) and were analyzed for their sensitivity to IFN-α-2b, as described above.
    Figure Legend Snippet: Effect of interferon (IFN)-α-2b on virus growth. A , Reduction of virus yield in the presence of antiviral activity. Vero E6 cells were infected with ∼100 pfu of the severe acute respiratory syndrome-related coronavirus (SARS CoV). At 1 h after infection, the supernatants were replaced with Dulbecco's modified Eagle's medium (DMEM) or DMEM containing the indicated amounts of IFN-α-2b (IU/mL) for a total of 72 h. Growth of the SARS CoV isolate Tor2 was determined by plaquetitration assay from 3 independent experiments, and data are mean ± SD. B , Analysis of cell-to-cell spread of SARS CoV in the presence and absence of IFN-α-2b. Vero E6 cell monolayers were infected with 10-fold dilutions of the SARS CoV. After 1 h of virus adsorption, the inoculum was removed, and cell monolayers were overlaid with 0.9% low-meltingpoint agarose in DMEM (B2) or DMEM containing 5000 IU/mL of IFNa- 2b (B1). On day 3 after infection, cells were stained with crystal violet. C , Protein analysis. Cell lysates were subjected to 10% SDS-PAGE. Protein was electrotransferred to polyvinylidene difluoride membrane (Immobilon P membrane; Millipore). Viral antigen was detected by use of a patient serum sample and horseradish peroxidase-conjugated anti-human IgG, by use of enhanced chemiluminescence. NP, nucleoprotein. D , Comparison of the effect of antiviral activity on different SARS CoV isolates. Vero E6 cells were infected with 4 independently isolated SARS CoVs (isolates Tor2, Tor3, Tor7, and Tor684) and were analyzed for their sensitivity to IFN-α-2b, as described above.

    Techniques Used: Activity Assay, Infection, Modification, Adsorption, Staining, SDS Page, Isolation

    Cytopathic effect in severe acute respiratory syndrome-related coronavirus (SARS CoV)-infected Vero E6 cells. Vero E6 cells were infected with the SARS CoV at an MOI of 0.001 for 1 h and then were incubated in Dulbecco's modified Eagle's medium containing the indicated amounts of interferon-α-2b (IU/mL). Cells were fixed with 4% formalin on day 3 after infection.
    Figure Legend Snippet: Cytopathic effect in severe acute respiratory syndrome-related coronavirus (SARS CoV)-infected Vero E6 cells. Vero E6 cells were infected with the SARS CoV at an MOI of 0.001 for 1 h and then were incubated in Dulbecco's modified Eagle's medium containing the indicated amounts of interferon-α-2b (IU/mL). Cells were fixed with 4% formalin on day 3 after infection.

    Techniques Used: Infection, Incubation, Modification

    36) Product Images from "Anti-SARS-CoV immunity induced by a novel CpG oligodeoxynucleotide"

    Article Title: Anti-SARS-CoV immunity induced by a novel CpG oligodeoxynucleotide

    Journal: Clinical Immunology (Orlando, Fla.)

    doi: 10.1016/j.clim.2005.09.014

    BW001 stimulates human PBMCs to produce strong anti-SARS-CoV activity. (A) The supernatants of human PBMCs treated with BW001 or BW002 were used to protect Vero E6 cells from challenge of 10× TICD50 SARS-CoV (see Materials and methods ). The dose of the stimulator was 6 μg/ml. Each symbol represents mean OD values ± SD. Each curve represents anti-SARS-CoV activity induced with or without CpG ODN. The result showed that BW001 could strongly stimulate human PBMCs to produce anti-SARS-CoV activity. (B) IFN-α contents in the supernatants used for anti-SARS-CoV were determined by ELISA method. Each column represents level of IFN-α induced with or without CpG ODN. BW001 could stimulate human PBMCs (3 × 10 6 /ml) to produce 160 pg/ml IFN-α that was much high than that induced by BW002 and medium. (C) Anti-SARS-CoV activity of recombinant IFN-α2b. “U” represents the international unit of IFN-α2b. Data from one representative experiment of three are shown.
    Figure Legend Snippet: BW001 stimulates human PBMCs to produce strong anti-SARS-CoV activity. (A) The supernatants of human PBMCs treated with BW001 or BW002 were used to protect Vero E6 cells from challenge of 10× TICD50 SARS-CoV (see Materials and methods ). The dose of the stimulator was 6 μg/ml. Each symbol represents mean OD values ± SD. Each curve represents anti-SARS-CoV activity induced with or without CpG ODN. The result showed that BW001 could strongly stimulate human PBMCs to produce anti-SARS-CoV activity. (B) IFN-α contents in the supernatants used for anti-SARS-CoV were determined by ELISA method. Each column represents level of IFN-α induced with or without CpG ODN. BW001 could stimulate human PBMCs (3 × 10 6 /ml) to produce 160 pg/ml IFN-α that was much high than that induced by BW002 and medium. (C) Anti-SARS-CoV activity of recombinant IFN-α2b. “U” represents the international unit of IFN-α2b. Data from one representative experiment of three are shown.

    Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay, Recombinant

    37) Product Images from "An adaptive compromise - Conflicting evolutionary pressures on arthropod-borne Zika virus dinucleotide composition in mammalian hosts and mosquito vectors"

    Article Title: An adaptive compromise - Conflicting evolutionary pressures on arthropod-borne Zika virus dinucleotide composition in mammalian hosts and mosquito vectors

    Journal: bioRxiv

    doi: 10.1101/2021.02.09.430415

    Infection of Aedes aegypti with ZIKV mutants. Aedes mosquitoes were offered an infectious bloodmeal containing equal RNA concentrations of each of the Zika viruses. Fully engorged females were selected and 14 days later subjected to a salivation assay (A) . Mosquito body homogenates (B) and mosquito saliva (C) were inoculated on Vero E6 cells to detect the presence of infectious virus. Bars represent the percentage of ZIKV positive samples with n between 21 and 36 engorged mosquitoes per experimental group. Asterisks represent significant difference from WT (Fisher exact test, P
    Figure Legend Snippet: Infection of Aedes aegypti with ZIKV mutants. Aedes mosquitoes were offered an infectious bloodmeal containing equal RNA concentrations of each of the Zika viruses. Fully engorged females were selected and 14 days later subjected to a salivation assay (A) . Mosquito body homogenates (B) and mosquito saliva (C) were inoculated on Vero E6 cells to detect the presence of infectious virus. Bars represent the percentage of ZIKV positive samples with n between 21 and 36 engorged mosquitoes per experimental group. Asterisks represent significant difference from WT (Fisher exact test, P

    Techniques Used: Infection

    Replication of ZIKV with increased UpA and CpG dinucleotides is restricted in vertebrate cell culture. Vertebrate Vero E6 cells (A) , A549 cells (B) and A549 ZAP knockout cells (C) were infected with the mutant ZIKV viruses at 1 RNA / cell. At the indicated days post infection (dpi) the 50% tissue culture infectious dose was determined by end point dilution assays. (D) Relative replication of mutant viruses was calculated by dividing mutant over WT virus titres at 2dpi. Data points represent the average of a total of four independent experiments, compiled of two experiments performed for each of two independently rescued virus populations. The error bars indicate one standard error of the mean. Single asterisks highlight significant differences in wildtype vertebrate cells from WT ZIKV and two asterisks the difference between A549 cells and A549 ZAP knockout cells (two-way ANOVA, FDR adjusted P
    Figure Legend Snippet: Replication of ZIKV with increased UpA and CpG dinucleotides is restricted in vertebrate cell culture. Vertebrate Vero E6 cells (A) , A549 cells (B) and A549 ZAP knockout cells (C) were infected with the mutant ZIKV viruses at 1 RNA / cell. At the indicated days post infection (dpi) the 50% tissue culture infectious dose was determined by end point dilution assays. (D) Relative replication of mutant viruses was calculated by dividing mutant over WT virus titres at 2dpi. Data points represent the average of a total of four independent experiments, compiled of two experiments performed for each of two independently rescued virus populations. The error bars indicate one standard error of the mean. Single asterisks highlight significant differences in wildtype vertebrate cells from WT ZIKV and two asterisks the difference between A549 cells and A549 ZAP knockout cells (two-way ANOVA, FDR adjusted P

    Techniques Used: Cell Culture, Knock-Out, Infection, Mutagenesis

    38) Product Images from "A human microsatellite DNA-mimicking oligodeoxynucleotide with CCT repeats negatively regulates TLR7/9-mediated innate immune responses via selected TLR pathways"

    Article Title: A human microsatellite DNA-mimicking oligodeoxynucleotide with CCT repeats negatively regulates TLR7/9-mediated innate immune responses via selected TLR pathways

    Journal: Clinical Immunology (Orlando, Fla.)

    doi: 10.1016/j.clim.2009.11.009

    ODN-induced inhibition of RNA and DNA-mediated antiviral activity in vitro. Human PBMCs were stimulated with inactivated Flu virus (PR8) or DNA virus (HSV-1) for 48 h, and the supernatants were serially diluted for protecting Vero E6 cells from VSV challenge. The anti-VSV activity was paralleled with that displayed by recombinant IFN-α. (a and e) Inactivated virus-mediated anti-VSV activity: (a) Flu virus (PR8) and (e) DNA virus (HSV-1). (b and f) ODN-induced inhibition of inactivated virus-mediated anti-VSV activity. Human PBMCs were cultured in medium containing inactivated PR8 (b) or HSV-1 (f) with or without different ODNs (MS19, IRS954, A151, and SAT05f) for 48 h, and the supernatants were harvested for testing their VSV protection effect. Each symbol represents PBMC from one of seven donors. (c and d) Dose–effect curves of ODNs on inhibiting RNA-induced anti-VSV activity. Human PBMCs were cultured in medium containing inactivated PR8 (c) or Imiquimod (d) with or without different dosages of MS19 or SAT05f for 48 h, and the supernatants were harvested for assaying their VSV protection effect. Representative data from one of three donors are shown. (g and h) Dose–effect curves of ODNs on inhibiting DNA-induced anti-VSV activity. Human PBMCs were cultured in medium containing inactivated HSV-1 (g) or A-class CpG ODN (2216) (h) with or without different dosages of MS19 or SAT05f for 48 h and the supernatants were harvested for assaying their VSV protection effect. Representative data from one of three donors are shown. One symbol represents one donor-derived sample. ⁎ P
    Figure Legend Snippet: ODN-induced inhibition of RNA and DNA-mediated antiviral activity in vitro. Human PBMCs were stimulated with inactivated Flu virus (PR8) or DNA virus (HSV-1) for 48 h, and the supernatants were serially diluted for protecting Vero E6 cells from VSV challenge. The anti-VSV activity was paralleled with that displayed by recombinant IFN-α. (a and e) Inactivated virus-mediated anti-VSV activity: (a) Flu virus (PR8) and (e) DNA virus (HSV-1). (b and f) ODN-induced inhibition of inactivated virus-mediated anti-VSV activity. Human PBMCs were cultured in medium containing inactivated PR8 (b) or HSV-1 (f) with or without different ODNs (MS19, IRS954, A151, and SAT05f) for 48 h, and the supernatants were harvested for testing their VSV protection effect. Each symbol represents PBMC from one of seven donors. (c and d) Dose–effect curves of ODNs on inhibiting RNA-induced anti-VSV activity. Human PBMCs were cultured in medium containing inactivated PR8 (c) or Imiquimod (d) with or without different dosages of MS19 or SAT05f for 48 h, and the supernatants were harvested for assaying their VSV protection effect. Representative data from one of three donors are shown. (g and h) Dose–effect curves of ODNs on inhibiting DNA-induced anti-VSV activity. Human PBMCs were cultured in medium containing inactivated HSV-1 (g) or A-class CpG ODN (2216) (h) with or without different dosages of MS19 or SAT05f for 48 h and the supernatants were harvested for assaying their VSV protection effect. Representative data from one of three donors are shown. One symbol represents one donor-derived sample. ⁎ P

    Techniques Used: Inhibition, Activity Assay, In Vitro, Recombinant, Cell Culture, Derivative Assay

    39) Product Images from "MERS-CoV pathogenesis and antiviral efficacy of licensed drugs in human monocyte-derived antigen-presenting cells"

    Article Title: MERS-CoV pathogenesis and antiviral efficacy of licensed drugs in human monocyte-derived antigen-presenting cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0194868

    Antiviral activity of three compounds on MERS-CoV infection in MDMs and immature MDDCs. (A) Monocyte-derived macrophages (MDMs), (B) immature dendritic cells (MDDCs) and (C) Vero E6 cells were pretreated with toremifene (TOMF), chloroquine (CQ), and chlorpromazine (CPZ) for 1 h followed by inoculation with MERS-CoV Jordan variant at an MOI = 0.1. Supernatants collected at 72 h post infection (hpi) for APCs and 48 hpi for Vero E6 cells were inoculated onto Vero E6 cells pre-seeded overnight in 96 well plates and incubated for 6 days. Wells with cytopathic effects were counted, and TCID 50 was determined using the Reed-Muench method. Cytotoxicity was measured in parallel using luminescent cell viability assay at 48 h after compounds addition to the mock-infected MDMs and immature MDDCs. Antiviral activity is shown in gray and cytotoxicity is shown in orange. Results are representative of 2 individual experiments with 3 replicates in each (means ± standard error of the means [SEM]).
    Figure Legend Snippet: Antiviral activity of three compounds on MERS-CoV infection in MDMs and immature MDDCs. (A) Monocyte-derived macrophages (MDMs), (B) immature dendritic cells (MDDCs) and (C) Vero E6 cells were pretreated with toremifene (TOMF), chloroquine (CQ), and chlorpromazine (CPZ) for 1 h followed by inoculation with MERS-CoV Jordan variant at an MOI = 0.1. Supernatants collected at 72 h post infection (hpi) for APCs and 48 hpi for Vero E6 cells were inoculated onto Vero E6 cells pre-seeded overnight in 96 well plates and incubated for 6 days. Wells with cytopathic effects were counted, and TCID 50 was determined using the Reed-Muench method. Cytotoxicity was measured in parallel using luminescent cell viability assay at 48 h after compounds addition to the mock-infected MDMs and immature MDDCs. Antiviral activity is shown in gray and cytotoxicity is shown in orange. Results are representative of 2 individual experiments with 3 replicates in each (means ± standard error of the means [SEM]).

    Techniques Used: Activity Assay, Infection, Derivative Assay, Variant Assay, Incubation, Endpoint Dilution Assay, Cell Viability Assay

    Related Articles

    Titration:

    Article Title: Characterization of cells susceptible to SARS-COV-2 and methods for detection of neutralizing antibody by focus forming assay
    Article Snippet: Using both Vero CCL-81 (ATCC® CCL-81™, referred to in this text as Vero WHO) and Vero E6 (Vero 1008, ATCC® CRL-1586™) cell lines, we determined if differences in foci number or size occurred to decide if one cell line was superior for titration by FFA ( – ). .. Although many laboratories utilize Vero E6 cells for viral titer measurements of SARS-CoV-2 [ , ], in our laboratory, Vero E6 cells typically resulted in about two-fold lower foci formation relative to Vero WHO cells ( ). is a representative image of an FFA showing the viral titration on both the Vero E6 and WHOs for both ~50 FFU and ~200 FFU when identical numbers of cells are seeded per well. ..

    Modification:

    Article Title: Analysis of N-Linked Glycosylation of Hantaan Virus Glycoproteins and the Role of Oligosaccharide Side Chains in Protein Folding and Intracellular Trafficking
    Article Snippet: Two endoplasmic reticulum (ER) chaperones, calnexin (CNX) and calreticulin (CRT), were found associated with the glycoproteins and presumably play a role in HTNV glycoprotein folding. .. HeLaT4+ cells and Vero E6 cells (ATCC C1008) were grown in Dulbecco's modified Eagles's medium containing 10% fetal bovine serum (FBS). .. BSR-T7 cells, which stably express T7 RNA polymerase , were kindly provided by K. K. Conzelmann (Max-von-Pettenkofer Institut, Munich, Germany) and were grown in Glasgow modified Eagle's medium containing 10% FBS. vTF7-3, a recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase , was a gift from B. Moss, National Institutes of Health, Bethesda, Md. vT-HTN M, a recombinant vaccinia virus that expresses HTNV glycoproteins (Gn and Gc), was constructed in this laboratory (unpublished data).

    Article Title: N-Glycans on the Rift Valley Fever Virus Envelope Glycoproteins Gn and Gc Redundantly Support Viral Infection via DC-SIGN
    Article Snippet: Media, Cells, and Viruses Minimum Essential Medium (MEM)-alpha supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA, USA), penicillin (100 U/mL), streptomycin (100 µg/mL), and hygromycin B (600 µg/mL) was used to maintain BHK/T7-9 cells that express the T7 RNA polymerase [ ]. .. Vero E6 cells (ATCC C1008) were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL). .. A Jurkat parental cell line and Jurkat cells stably expressing human DC-SIGN (Jurkat-DC-SIGN) or L-SIGN (Jurkat-L-SIGN) were kindly provided by Dr. Rafael Delgado (Molecular Microbiology Laboratory, Hospital Universitario 12 de Octubre, Madrid, Spain) [ ].

    Article Title: Novel Paju Apodemus Paramyxovirus 1 and 2, Harbored by Apodemus agrarius in The Republic of Korea
    Article Snippet: Cell lines Vero E6 cells (ATCC, #DR-L2785), human lung adenocarcinoma cells (A549) (ATCC, #CCL-185), and human umbilical vein endothelial cells (HUVEC) (ATCC, #CRL1730) were purchased from ATCC. .. Vero E6, A549, and HUVEC were cultured in DMEM supplemented with Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum, 1 mM sodium pyruvate, 2 mL L-glutamine, and 50 mg/ml gentamicin. .. The cultures were incubated at 37°C in a 5% CO2 incubator until use.

    Article Title: Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus
    Article Snippet: Thus, rMP12-PTNSs is an alternative candidate vaccine which can be amplified in MRC-5 cells and encodes a negative DIVA marker. .. Media, cells and viruses VeroE6 cells (ATCC CRL-1586), 293 cells (ATCC CRL-1573), MRC-5 cells (ATCC CCL-171) and MEF cells were maintained in Dulbecco's modified minimum essential medium (DMEM) containing 10% fetal calf serum (FCS). .. BHK/T7-9 cells that stably express T7 RNA polymerase were maintained in MEM-alpha containing 10% FCS with 600 µg/ml of hygromycin.

    Article Title: Mapping the Golgi Targeting and Retention Signal of Bunyamwera Virus Glycoproteins
    Article Snippet: .. HeLa T4+ cells ( ) and Vero E6 (ATCC C1008) cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. .. A recombinant vaccinia virus, vTF7-3, which synthesizes bacteriophage T7 RNA polymerase , was a gift from Bernard Moss (National Institutes of Health, Bethesda, Md.).

    Article Title: Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)
    Article Snippet: The number of living epimastigotes and metacyclic trypomastigotes was then determined by cell counting via a Neubauer chamber after 24 h, 48 h and 72 h. The metacyclic trypomastigotes were purified from the 72 h-old cultures by DEAE-51 cellulose chromatography ( ). .. To obtain amastigote forms, metacyclic trypomastigotes derived in vitro were used to infect Vero cells (ATCC CRL-1586), which were then cultivated in Dulbecco's Modified Eagle's medium (Sigma) supplemented with 5% FBS and incubated in a humidified atmosphere with 5% CO2 at 37°C. .. After 10 days of infection, the amastigotes released into the supernatant were harvested by centrifugation at 1,000 g for 5 min. L. major promastigotes were grown at 28°C in Schneider's medium (Sigma) supplemented with 10% FBS and 10 mg/L haemin.

    Cell Culture:

    Article Title: Novel Paju Apodemus Paramyxovirus 1 and 2, Harbored by Apodemus agrarius in The Republic of Korea
    Article Snippet: Cell lines Vero E6 cells (ATCC, #DR-L2785), human lung adenocarcinoma cells (A549) (ATCC, #CCL-185), and human umbilical vein endothelial cells (HUVEC) (ATCC, #CRL1730) were purchased from ATCC. .. Vero E6, A549, and HUVEC were cultured in DMEM supplemented with Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum, 1 mM sodium pyruvate, 2 mL L-glutamine, and 50 mg/ml gentamicin. .. The cultures were incubated at 37°C in a 5% CO2 incubator until use.

    Derivative Assay:

    Article Title: Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)
    Article Snippet: The number of living epimastigotes and metacyclic trypomastigotes was then determined by cell counting via a Neubauer chamber after 24 h, 48 h and 72 h. The metacyclic trypomastigotes were purified from the 72 h-old cultures by DEAE-51 cellulose chromatography ( ). .. To obtain amastigote forms, metacyclic trypomastigotes derived in vitro were used to infect Vero cells (ATCC CRL-1586), which were then cultivated in Dulbecco's Modified Eagle's medium (Sigma) supplemented with 5% FBS and incubated in a humidified atmosphere with 5% CO2 at 37°C. .. After 10 days of infection, the amastigotes released into the supernatant were harvested by centrifugation at 1,000 g for 5 min. L. major promastigotes were grown at 28°C in Schneider's medium (Sigma) supplemented with 10% FBS and 10 mg/L haemin.

    In Vitro:

    Article Title: Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)
    Article Snippet: The number of living epimastigotes and metacyclic trypomastigotes was then determined by cell counting via a Neubauer chamber after 24 h, 48 h and 72 h. The metacyclic trypomastigotes were purified from the 72 h-old cultures by DEAE-51 cellulose chromatography ( ). .. To obtain amastigote forms, metacyclic trypomastigotes derived in vitro were used to infect Vero cells (ATCC CRL-1586), which were then cultivated in Dulbecco's Modified Eagle's medium (Sigma) supplemented with 5% FBS and incubated in a humidified atmosphere with 5% CO2 at 37°C. .. After 10 days of infection, the amastigotes released into the supernatant were harvested by centrifugation at 1,000 g for 5 min. L. major promastigotes were grown at 28°C in Schneider's medium (Sigma) supplemented with 10% FBS and 10 mg/L haemin.

    Incubation:

    Article Title: Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)
    Article Snippet: The number of living epimastigotes and metacyclic trypomastigotes was then determined by cell counting via a Neubauer chamber after 24 h, 48 h and 72 h. The metacyclic trypomastigotes were purified from the 72 h-old cultures by DEAE-51 cellulose chromatography ( ). .. To obtain amastigote forms, metacyclic trypomastigotes derived in vitro were used to infect Vero cells (ATCC CRL-1586), which were then cultivated in Dulbecco's Modified Eagle's medium (Sigma) supplemented with 5% FBS and incubated in a humidified atmosphere with 5% CO2 at 37°C. .. After 10 days of infection, the amastigotes released into the supernatant were harvested by centrifugation at 1,000 g for 5 min. L. major promastigotes were grown at 28°C in Schneider's medium (Sigma) supplemented with 10% FBS and 10 mg/L haemin.

    other:

    Article Title: Anti-SARS-CoV-2 Potential of Artemisinins In Vitro
    Article Snippet: Cells and Virus Vero E6 cells (ATCC, no. 1586) were grown and maintained in minimum Eagle’s medium (Gibco Invitrogen) supplemented with 10% fetal bovine serum (Gibco Invitrogen) at 37 °C in 5% CO2 .

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    ATCC vero e6 cells
    Pre-treatment of cells with emodin and berberine. Graphic of relative foci number after treatment of <t>Vero</t> <t>E6</t> cells with berberine or emodin. Cells treated with drug diluent were used as negative control. Results are the means (±SD) from three independent experiments and are expressed as relative values compared to untreated cells. *** p
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    Pre-treatment of cells with emodin and berberine. Graphic of relative foci number after treatment of Vero E6 cells with berberine or emodin. Cells treated with drug diluent were used as negative control. Results are the means (±SD) from three independent experiments and are expressed as relative values compared to untreated cells. *** p

    Journal: Viruses

    Article Title: Natural Products Isolated from Oriental Medicinal Herbs Inactivate Zika Virus

    doi: 10.3390/v11010049

    Figure Lengend Snippet: Pre-treatment of cells with emodin and berberine. Graphic of relative foci number after treatment of Vero E6 cells with berberine or emodin. Cells treated with drug diluent were used as negative control. Results are the means (±SD) from three independent experiments and are expressed as relative values compared to untreated cells. *** p

    Article Snippet: Berberine reduced virus infectivity by almost 80% in Vero E6 cells.

    Techniques: Negative Control

    Viability of emodin- and berberine-treated Vero E6 cells. Cells were incubated with different concentrations of emodin ( A ) or berberine ( B ) and cell viability was evaluated after 24 h, 48 h, and 72 h.

    Journal: Viruses

    Article Title: Natural Products Isolated from Oriental Medicinal Herbs Inactivate Zika Virus

    doi: 10.3390/v11010049

    Figure Lengend Snippet: Viability of emodin- and berberine-treated Vero E6 cells. Cells were incubated with different concentrations of emodin ( A ) or berberine ( B ) and cell viability was evaluated after 24 h, 48 h, and 72 h.

    Article Snippet: Berberine reduced virus infectivity by almost 80% in Vero E6 cells.

    Techniques: Incubation

    Replication of rMP12-PTNSs and rMP12-SFSNSs in cell culture. (A) VeroE6 cells, (B) MEF cells or (C) MRC-5 cells were mock-infected or infected with MP-12, rMP12-C13type, rMP12-PTNSs, or rMP12-SFSNSs at a m.o.i of 0.01. Culture supernatants were collected at 72 hpi (A and B), or indicated time points (C) and virus titer was determined by plaque assay with VeroE6 cells. Means+standard deviations of three independent experiments are shown in the graph. Asterisk represents statistical significance (Unpaired t-test, **p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus

    doi: 10.1371/journal.pntd.0002181

    Figure Lengend Snippet: Replication of rMP12-PTNSs and rMP12-SFSNSs in cell culture. (A) VeroE6 cells, (B) MEF cells or (C) MRC-5 cells were mock-infected or infected with MP-12, rMP12-C13type, rMP12-PTNSs, or rMP12-SFSNSs at a m.o.i of 0.01. Culture supernatants were collected at 72 hpi (A and B), or indicated time points (C) and virus titer was determined by plaque assay with VeroE6 cells. Means+standard deviations of three independent experiments are shown in the graph. Asterisk represents statistical significance (Unpaired t-test, **p

    Article Snippet: Media, cells and viruses VeroE6 cells (ATCC CRL-1586), 293 cells (ATCC CRL-1573), MRC-5 cells (ATCC CCL-171) and MEF cells were maintained in Dulbecco's modified minimum essential medium (DMEM) containing 10% fetal calf serum (FCS).

    Techniques: Cell Culture, Infection, Plaque Assay

    Generation of rMP12-PTNSs and rMP12-SFSNSs. (A) Schematics of MP-12 S-segments encoding mutation or foreign gene in place of MP-12 NSs. The rMP12-C13type (C13type) lacks 69% of the NSs ORF as described previously [44] . The rMP12-PTNSs, and rMP12-SFSNSs encode NSs of Punta Toro virus Adames strain and Sandfly fever Sicilian virus, respectively. The expected phenotype corresponding to each S-segment is also presented. (B) Plaque phenotypes of MP-12, rMP12-PTNSs and rMP12-SFSNSs at 4 dpi. Plaque assay was performed with VeroE6 cells overlaid with 0.6% noble agar and stained with Neutral red.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus

    doi: 10.1371/journal.pntd.0002181

    Figure Lengend Snippet: Generation of rMP12-PTNSs and rMP12-SFSNSs. (A) Schematics of MP-12 S-segments encoding mutation or foreign gene in place of MP-12 NSs. The rMP12-C13type (C13type) lacks 69% of the NSs ORF as described previously [44] . The rMP12-PTNSs, and rMP12-SFSNSs encode NSs of Punta Toro virus Adames strain and Sandfly fever Sicilian virus, respectively. The expected phenotype corresponding to each S-segment is also presented. (B) Plaque phenotypes of MP-12, rMP12-PTNSs and rMP12-SFSNSs at 4 dpi. Plaque assay was performed with VeroE6 cells overlaid with 0.6% noble agar and stained with Neutral red.

    Article Snippet: Media, cells and viruses VeroE6 cells (ATCC CRL-1586), 293 cells (ATCC CRL-1573), MRC-5 cells (ATCC CCL-171) and MEF cells were maintained in Dulbecco's modified minimum essential medium (DMEM) containing 10% fetal calf serum (FCS).

    Techniques: Mutagenesis, Plaque Assay, Staining

    Degradation of PKR in VeroE6 cells infected with rMP12-PTNSs or rMP12-SFSNSs. (A) VeroE6 cells were mock-infected or infected with MP-12, rMP12-C13type, rMP12-PTNSs or rMP12-SFSNSs at a m.o.i of 3, and cells were collected at 16 hpi. Western blot was performed with anti-PKR, anti-RVFV and anti-β-actin antibodies. (B) VeroE6 cells were mock-infected or infected with rMP12-NSs-Flag [32] , rMP12-C13type, rMP12-PTNSs-Flag or rMP12-SFSNSs-Flag at a m.o.i of 3, and Western blot was performed as described above. Anti-Flag antibody was used for the detection of NSs-Flag. Representative data from three independent experiments are shown.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus

    doi: 10.1371/journal.pntd.0002181

    Figure Lengend Snippet: Degradation of PKR in VeroE6 cells infected with rMP12-PTNSs or rMP12-SFSNSs. (A) VeroE6 cells were mock-infected or infected with MP-12, rMP12-C13type, rMP12-PTNSs or rMP12-SFSNSs at a m.o.i of 3, and cells were collected at 16 hpi. Western blot was performed with anti-PKR, anti-RVFV and anti-β-actin antibodies. (B) VeroE6 cells were mock-infected or infected with rMP12-NSs-Flag [32] , rMP12-C13type, rMP12-PTNSs-Flag or rMP12-SFSNSs-Flag at a m.o.i of 3, and Western blot was performed as described above. Anti-Flag antibody was used for the detection of NSs-Flag. Representative data from three independent experiments are shown.

    Article Snippet: Media, cells and viruses VeroE6 cells (ATCC CRL-1586), 293 cells (ATCC CRL-1573), MRC-5 cells (ATCC CCL-171) and MEF cells were maintained in Dulbecco's modified minimum essential medium (DMEM) containing 10% fetal calf serum (FCS).

    Techniques: Infection, Western Blot

    Multistep growth curve of recombinant hMPVs carrying mutations in the phosphorylation site. Vero E6 cells in 35-mm dishes were infected with each recombinant hMPV at an MOI of 0.01. After adsorption for 1 h, the inocula were removed and the infected cells were washed 3 times with Opti-MEM. Fresh Opti-MEM containing 2% FBS then was added and cells were incubated at 37°C for various time periods. Aliquots of the cell culture fluid were removed at the indicated intervals. Viral titer was determined by an immunostaining assay in Vero E6 cells. Viral titers of each mutant at each time point were compared to those of rhMPV (*, P

    Journal: Journal of Virology

    Article Title: Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis

    doi: 10.1128/JVI.00755-16

    Figure Lengend Snippet: Multistep growth curve of recombinant hMPVs carrying mutations in the phosphorylation site. Vero E6 cells in 35-mm dishes were infected with each recombinant hMPV at an MOI of 0.01. After adsorption for 1 h, the inocula were removed and the infected cells were washed 3 times with Opti-MEM. Fresh Opti-MEM containing 2% FBS then was added and cells were incubated at 37°C for various time periods. Aliquots of the cell culture fluid were removed at the indicated intervals. Viral titer was determined by an immunostaining assay in Vero E6 cells. Viral titers of each mutant at each time point were compared to those of rhMPV (*, P

    Article Snippet: When Vero E6 cells were infected at a higher MOI (0.1), the highest titer of rhMPV, rhMPV-S57A, rhMPV-S60A, and rhMPV-S57A-S60A reached 106.7 , 106.5 , 106.3 , and 105.8 , respectively (data not shown).

    Techniques: Recombinant, Infection, Adsorption, Incubation, Cell Culture, Immunostaining, Mutagenesis

    Recovery of recombinant hMPVs carrying mutations in the phosphorylation sites of M2-1. (A) Immunostaining spots formed by recombinant hMPVs. Vero E6 cells were infected with recombinant hMPV mutants and incubated at 37°C for 5 days. The cells were stained with an anti-hMPV N protein monoclonal antibody. (B) Plaque morphology of recombinant hMPVs. An agarose overlay plaque assay was performed on a monolayer of Vero E6 cells. Viral plaques were developed at day 8 postinfection. The plaque pictures were taken from different dilutions of each hMPV mutant. Data were averages from 20 plaques ± 1 standard deviation.

    Journal: Journal of Virology

    Article Title: Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis

    doi: 10.1128/JVI.00755-16

    Figure Lengend Snippet: Recovery of recombinant hMPVs carrying mutations in the phosphorylation sites of M2-1. (A) Immunostaining spots formed by recombinant hMPVs. Vero E6 cells were infected with recombinant hMPV mutants and incubated at 37°C for 5 days. The cells were stained with an anti-hMPV N protein monoclonal antibody. (B) Plaque morphology of recombinant hMPVs. An agarose overlay plaque assay was performed on a monolayer of Vero E6 cells. Viral plaques were developed at day 8 postinfection. The plaque pictures were taken from different dilutions of each hMPV mutant. Data were averages from 20 plaques ± 1 standard deviation.

    Article Snippet: When Vero E6 cells were infected at a higher MOI (0.1), the highest titer of rhMPV, rhMPV-S57A, rhMPV-S60A, and rhMPV-S57A-S60A reached 106.7 , 106.5 , 106.3 , and 105.8 , respectively (data not shown).

    Techniques: Recombinant, Immunostaining, Infection, Incubation, Staining, Plaque Assay, Mutagenesis, Standard Deviation